Circadian expression of the light-harvesting protein of Photosystem II in etiolated bean leaves following a single red light pulse: Coordination with the capacity of the plant to form chlorophyll and the thylakoid-bound protease

The appearance of the light harvesting II (LHC II) protein in etiolated bean leaves, as monitored by immunodetection in LDS-solubilized leaf protein extracts, is under phytochrome control. A single red light pulse induces accumulation of the protein, in leaves kept in the dark thereafter, which follows circadian oscillations similar to those earlier found for Lhcb mRNA (Tavladoraki et al. (1989) Plant Physiol 90: 665–672). These oscillations are closely followed by oscillations in the capacity of the leaf to form Chlorophyll (Chl) in the light, suggesting that the synthesis of the LHC II protein and its chromophore are in close coordination. Experiments with levulinic acid showed that PChl(ide) resynthesis does not affect the LHC II level nor its oscillations, but new Chl a synthesis affects LHC II stabilization in thylakoids, implicating a proteolytic mechanism. A proteolytic activity against exogenously added LHC II was detected in thylakoids of etiolated bean leaves, which was enhanced by the light pulse. The activity, also under phytochrome control, was found to follow circadian oscillations in verse to those in the stabilization of LHC II protein in thylakoids. Such a proteolytic mechanism therefore, may account for the circadian changes observed in LHC II protein level, being implicated in pigment-protein complex assembly/stabilization during thylakoid biogenesis.Abbreviations Chl chlorophyll - CL continuous light - D dark - FR far-red light - LA levulinic acid - LHC II light-harvesting complex serving Photosystem II - PChl(ide) protochlorophyllide - PCR protochlorophyllide oxidoreductase - R red light     

Studies on Greening of Etiolated Seedlings

Evidence is given that a selective light-pretreatment of the embryonic axis exerts a deep influence on the greening in primary leaves of 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Limburg). After a subsequent dark incubation of sufficient length and a final exposure of the entire plants to continuous illumination the lag phase of chlorophyll synthesis is completely removed. In particular the highly meristematic hook tissue seems to be responsible for this light effect. Lengthening of the dark period following pre-irradiation increased the capability of chlorophyll production in the main white light period, reaching its maximum after about 12 hours of darkness. The period of dark incubation for elimination of the lag phase is considerably longer in plants with shielded leaves than the length of the lag phase in etiolated seedlings of the same age, exposed entirely to continuous light. This difference may be explained by the synergistic effect between leaves and embryonic axis. Evidence for this interorgan cooperation is given by experiments with a selective light-pretreatment of leaves and embryonic axis. After a 5 min pre-exposure to white light of whole plants the leaves of some of the plants were shielded and these plants received a further pre-illumination of 2 hours on their embryonic axis. In all the pre-irradiated, etiolated plants the lag phase of chlorophyll synthesis was eliminated during the main white light period, following a dark incubation of 2 hours. Additional and preferential light activation of the embryonic axis during the pretreatment had no significant effect on chlorophyll production during the white light illumination after a 2 hours dark incubation, but resulted in a lower yield of chlorophylls after 18 hours dark incubation compared to the white light controls, receiving no selective light-pretreatment on the embryonic axis. From our results we can decisively conclude that a simultaneous light-pretreatment of both, leaves and embryonic axis, is more effective and beneficial for building up a capacity of chlorophyll synthesis in the leaves than either a selective light-pretreatment of the embryonic axis alone or a simultaneous pre-illumination of leaves and embryonic axis, immediately followed by an additional preirradiation of the embryonic axis. Therefore, we think that several photoactive sites are involved in de-etiolation processes of intact, etiolated seedings. Light activation of the embryonic axis stimulates the development of this organ and contributes to the greening processes in the leaf. At the same time, by irradiating the leaf, light activates the photo-sensitive site in the leaf itself, which also develops a capacity for chlorophyll synthesis. Both photo-acts are cooperative, explaining the enhanced chlorophyll production. Additional pre-irradiation of the embryonic axis after a short illumination of whole plants favours its own development and reduces the synthetic capacity of the leaf. A prolonged far-red pretreatment induces qualitatively the same response as white light. We assume that these effects on lag phase removal and chlorophyll production, induced in etiolated, primary bean leaves by selective irradiation of the embryonic axis, is a phytochrome-mediated process. Our results indicate a transmission of light-induced stimuli from one organ to another.     

Studies on Greening of Etiolated Seedlings II. Leaf Greening by Phytochrome Action in the Embryonic Axis

We could demonstrate that greening of primary bean leaves in etiolated seedlings of Phaseolus vulgaris cv. Limburg can be controlled by a selective light-pretreatment of the embryonic axis. This light-induced interorgan synergism proved to be a phytochrome-mediated process. The red/farred photoreversible effect on the embryonic axis seems to be primarily linked to changes in the energy metabolism of the primary leaves. Phototransformation of the protochlorophyll present and pigment synthesis are very dependent upon an adequate supply of biochemical energy. When the embryonic axis is selectively pre-exposed to red light for a short time, respiration is markedly enhanced in the leaves and photosynthesis starts immediately upon illumination of the etiolated leaves after an incubation period of optimal length in the dark. The stimulatory effect of the red pretreatment on leaf respiration and photosynthetic capacity could be abolished to the level of the dark controls by a subsequent far-red irradiation on the embryonic axis. It is therefore postulated that phytochrome plays a regulatory role in interorgan cooperation. The metabolic changes involved in photomorphogenesis of etiolated seedlings are closely related to changes in energy production. Our data indicate that the primary act of phytochrome becomes operative at the biochemical level by its directional influence on the energy balance of the cell and coordinates the use of metabolic energy within a tissue and between organs.     

Light-induced Polysome Formation in Etiolated Leaves: Kinetics of Inhibition by Antibiotics

Red light-induced, far red light-reversible increase in etiolated bean (Phaseolus vulgaris, var. Asgrow Valentine) leaf polyribosomes was shown to be sensitive to actinomycin D, cycloheximide, and rifampicin inhibition. Actinomycin prevented response to red light if administered simultaneously with a 10-minute illumination, had no immediate effect if given 2 hours after illumination, but was again rapidly inhibitory at 4 and 6 hours. The effects of actinomycin and far red light were more than additive.     

Gibberellic Acid effects on greening in pea seedlings

The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly.     

Regulation by light and metabolites of ferredoxin-dependent glutamate synthase in maize

The regulation of Fd-glutamate synthase (Fd-GOGAT, EC 1.4.1.7) and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) was investigated in maize ( Zea mays L. cv. DEA) (1) during development starting from 7- to 11-day-old seedlings, (2) by treatment of 7-day-old etiolated leaves with intermittent light pulses to activate (red) and inactivate (far-red) phytochromes and (3) in 7-day-old green leaves grown under 16-h light/8-h dark cycles. Fd-GOGAT mRNA accumulated 4-fold, and the enzyme polypeptide (3-fold) and activity (3-fold) also increased in leaf cells, while NADH-GOGAT activity remained constantly low. Leaf-specific induction of Fd-GOGAT mRNA (3-fold) occurred in etiolated leaves by low fluence red light, and far-red light reversibly repressed the mRNA accumulation. Red/far-red reversible induction also occurred for Fd-GOGAT polypeptide (2-fold) and activity (2-fold), implicating the phytochrome-dependent induction of Fd-GOGAT. In contrast, NADH-GOGAT activity remained constant, irrespective of red/far-red light treatments. Fd-GOGAT showed diurnal changes under light/dark cycles with the maximum early in the morning and the minimum in the afternoon at the levels of mRNA, enzyme polypeptide and activity. Gln diurnally changed in parallel with Fd-GOGAT mRNA. The induction of Fd-GOGAT provides evidence that light and metabolites are the major signal for the Gln and Glu formation in maize leaf cells.     

Phytochrome action on the timing of cell division in adiantum gametophytes

When filamentous protonemata of Adiantum capillus-veneris L. precultured under continuous red light were transferred to the dark, the apical cell divided about 24 to 36 hours thereafter. The time of the cell division was delayed for several hours by a brief exposure to far red light given before the dark incubation. The effect of far red light was reversed by a small dose of red light given immediately after the preceding far red light. The effects of red and far red light were repeatedly reversible, indicating that the timing of cell division was regulated by a phytochrome system. When a brief irradiation with blue light was given before the dark incubation, the cell division occurred after 17 to 26 hours in darkness. A similar red far red reversible effect was also observed in the timing of the blue light-induced cell division. Thus, the timing of cell division appeared to be controlled by phytochrome and a blue light-absorbing pigment.     

Interactions between Light and the Circadian Clock in the Regulation of CAT2 Expression in Arabidopsis

In Arabidopsis seedlings germinated and grown in continuous light, CAT2 mRNA abundance peaks 1 d after imbibition, consistent with the role of catalase in detoxifying H2O2 generated during the [beta]-oxidation of fatty acids stored in the seed. A second peak of CAT2 mRNA abundance, of lower amplitude than the initial peak, appears 6 d after imbibition and may be associated with the development of photosynthetic competence and induction of photorespiration. This second peak in steady-state CAT2 mRNA abundance is regulated by light and is not seen in etiolated seedlings. CAT2 mRNA accumulation is induced by exposure to high-fluence blue or far-red light but not by red light. In addition, light induction is unaffected by several mutations that block blue light-mediated inhibition of hypocotyl elongation (blu1, blu2, blu3, hy4), suggesting phytochrome involvement. When etiolated seedlings are transferred to continuous white light, CAT2 mRNA rapidly (within 30 min) accumulates. It is interesting that in these seedlings CAT2 mRNA abundance undergoes pronounced oscillations with a circadian (24 h) periodicity, indicating control by the endogenous circadian clock. No such oscillations are detected in CAT2 mRNA abundance in etiolated seedlings prior to illumination. Control of CAT2 expression by the circadian clock is also seen in 5-week-old plants grown in a light-dark cycle and transferred either to continuous dark or to continuous light; in continuous light the circadian oscillations in CAT2 mRNA abundance persist for at least five circadian cycles, indicating the robustness of this circadian rhythm.     

Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of δ-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Red Light-Independent Instability of Oat Phytochrome mRNA in Vivo

Phytochrome A (phyA) mRNA abundance decreased rapidly in total RNA samples isolated from 4-day-old etiolated oat seedlings following a red light pulse. Putative in vivo phyA mRNA degradation products were detectable both before and after red light treatment. Cordycepin-treated coleoptiles were unable to accumulate the chlorophyll a/b-binding protein mRNA in response to red light, indicating that cordycepin effectively inhibited mRNA synthesis. In cordycepin-treated coleoptiles, phyA mRNA rapidly decreased in abundance, consistent with the hypothesis that phyA mRNA is inherently unstable, rather than being destabilized after red light treatment of etiolated oat seedlings.     

Influence of Light on the Bioelectric Potential of the Bean (Phaseolus vulgaris) Hypocotyl Hook

By use of surface electrodes electropotenlial measurements were carried out on hypocotyl hooks of Phaseolus vulgaris seedlings. The hooks were illuminated with a small spot of white, blue, red or far red light. The potential changes in bean hypocotyl hooks do not show the red-far red reversible characteristics of phytochrome-mediated processes. By experimenting with inhibitors of photosynthesis we could demonstrate that the light-triggered potential changes in green bean hooks are correlated to photosynthetic electron transport phenomena. The red-light-induced transient is a depolarization, whereas blue light induces a hyperpolarization. Etiolated beans exhibit no bioelectric potential changes when subjected to red or far red irradiations. Blue light and white light induce a strong hyperpolarization in etiolated hooks cells. This transient seems to be an action potential induced by light. The action potential is influenced by inhibitors of electron transport and oxidative phosphorylation. By comparing the action spectrum of the action potential induced by light with the absorption spectra of extracted carotenoids and xanthophylls from etiolated bean hypocotyl hooks, we observed similarities.     

Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea mays L. Leaves

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10(-5)m and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions.     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Characterization of protoheme levels in etiolated and greening plant tissues

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.