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.     

Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light.     

White Light Effects on the mRNA for the Light-Harvesting Chlorophyll a/b-Protein in Lemna gibba L. G-3

Translation products of poly(A) mRNA isolated from Lemna gibba L. G-3 include a major polypeptide of 32,000 daltons which is immunoprecipitated by antiserum to chlorophyll a/b-protein from Chlamydomonas. This 32,000 dalton polypeptide represents a precursor to the light-harvesting chlorophyll a/b-protein of molecular weight 28,000 found in the thylakoid membranes of Lemna gibba. The amount of this translatable mRNA decreases relative to other translatable mRNAs when green plants grown in continuous white light are placed in darkness. This decrease occurs rapidly. The most rapid decline occurs during the first day; after 4 days of darkness, only a low level of this mRNA can be detected by in vitro translation. When the plants are returned to white light there is an increase in the relative level of this mRNA which can be easily detected within two hours. The in vivo synthesis of this protein has been assayed under the different light conditions. The light effects on the in vivo synthesis of the chlorophyll a/b-protein reflect the light effects on the translatable mRNA for the polypeptide. The results indicate that light induced changes in the synthesis, processing, or degradation of chlorophyll a/b-protein mRNA could account for the light-induced changes observed in the effective synthesis rates for the chlorophyll a/b-protein in vivo.     

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.     

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using ³²P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.     

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559_HP). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.     

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.     

Effect of Intermittent and Continuous Light on Chlorophyll Formation in Etiolated Plants

Short impulses of white light induce continuous synthesis of chlorophyll a and b in etiolated barley leaves. No lag phase is observed, and the rate of chlorophyll a accumulation is much higher than that of chlorophyll b, so that the a/b ratio is very high (12 to 20). The chlorophyll accumulation reaches a plateau at about 70 flashes after which the rate of their formation decreases appreciably. When the etiolated plants, after exposure to about 80 to 100 flashes, are transferred to continuous light, one can observe that the rate of formation of chlorophyll a and b increases during the first hour, and after that becomes still more rapid. At the same time the a/b ratio falls and it reaches a value of about 3 normally found in green leaves.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Involvement of Ethylene in Phytochrome-mediated Carotenoid Synthesis

Accumulation of carotenoid pigments in the shoot apex of etiolated pea (Pisum sativum cv. Alaska) seedlings is completely prevented by ethylene. Under certain conditions carotenoid synthesis is normally controlled by endogenously produced ethylene. The gas completely inhibits carotenoid synthesis induced either by continuous white light or brief illumination with red light, but only partially inhibits light-induced chlorophyll formation. Far red illumination followed by red illumination reverses the action of red light on carotenoid synthesis. Red light-induced carotenogenesis is partly or wholly caused by phytochrome-mediated inhibition of ethylene 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.

     

Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O₂ evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period.     

Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook

The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO₂ assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.

The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species.

     

Formation of photosynthetic pigments and quinones and development of photosynthetic activity in barley etioplasts during greening in intermittent and continuous white light

The appearance and development of photosynthetic activity, and the accumulation of chlorophylls, carotenoids and quinones, was investigated in etiolated barley shoots (Hordeum vulgare L. cv. Villa) during greening in flash light, periodic light-dark cycles, and continuous white light. Greening and the development of photosynthetic activity was delayed in flash and periodic light compared to continuous white light. Photosystem II activity occurred after 6 light-dark cycles and increased continuously during greening. After 3 h greening in continuous white light, photosystem II activity appeared with a very high rate and decreased to that of a green leaf after 50 h greening. Parallel to the development of photosynthetic activity, light stimulated the biosynthesis of prenyllipids. Moreover, chlorophylls and those carotenoids and quinones that are contained in etioplasts in relatively small amounts, were particularly enhanced in their biosynthesis. Chlorophyll a was synthesized without a lag phase during greening in flash light, whereas a 2 h lag phase occurred in continuous white light. In all three modes of illumination the formation of chlorophyll a exceeded that of chlorophyll b. After 4 flashes and 2 light-dark cycles, chlorophyll b could be detected with a very high initial a/b ratio. Higher chlorophyll a/b ratios were reached after 200 flashes (a/b=10.9) and 50 light-dark cycles (a/b=6.6) than after 50 h continuous white light (a/b=3.3). The formation of carotenes, lutein, violaxanthin and neoxanthin was also enhanced by light. This was also confirmed for plast-ouinone-9. ?-tocopherol,α-tocoquinone and phylloquinone. A comparison of the carotenoid and quinone composition of the differentiating thylakoid membrane before and after onset of photosynthesis, reveals that the photosynthetic membrane is already equipped with photosynthetic pigments and quinones before the appearance of photosystem II activity. It is concluded that during development of the photo-synthetic apparatus the thylakoid membrane with its structural and functional constituents is formed first. In a second and slower process the water splitting enzyme system and enzymes of the Calvin cycle are activated.     

Synthesis and turnover of the light-harvesting chlorophylla/b-protein inLemna gibba grown with intermittent red light: possible translational control

Lemna gibba L. G-3 plants grown heterotrophically in the dark with intermittent red light (2 min every 8 h) contain a substantial amount of translatable mRNA encoding the light-harvesting chlorophyll (Chl)a/b-protein. However, very little [³⁵S]methionine is incorporated into the apoproteins during a 1-h labeling period in the dark in these plants compared to plants grown in continuous white light. The Chla/b-protein mRNA is found to be associated with functioning polysomes in plants grown in the dark with intermittent red illumination (R plants). The small amounts of the apoproteins which are synthesized by these plants are found in the membrane fraction; neither the mature apoproteins nor their precursor(s) can be detected immunologically in the soluble fraction. The protein does not accumulate in these plants. Pulse-chase experiments with the R plants demonstrate that the newly synthesized apoproteins have a half-life of about 10 h in the dark. This turnover is not sufficient to explain the observed 20-fold difference in [³⁵S]methionine incorporation into the apoprotein between white-light-grown and R plants. We therefore suggest that the synthesis of the Chla/b-apoproteins can be regulated by a light-dependent step at the level of translation, and that this regulation occurs after the initiation of translation.Abbreviations Chl chlorophyll - W Lemna plants grown in continuous white light - R plants grown heterotrophically in the dark with intermittent red light (2 min/8 h)     

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.

     

The photochemical activities and electron carriers of developing barley leaves

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559_LP, b-563 and b-559_HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559_LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559_HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.