Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

Studies on chlorophyll formation in Chlorella protothecoides I. Enhancing effects of light and added {delta}-aminoIevulinic acid, and suppressive effect of glucose on chlorophyll formation

Light-induced formation of chlorophyll in "etiolated" cellsof Chlorella protothecoides was studied under various experimentalconditions, (i) Two different types of enhancing effect of lightwere demonstrated: a "long-term" effect lasting for many hoursafter a relatively short illumination of etiolated cells anda "short-term" effect disappearing in a few hours after illumination,(ii) Addition of ALA caused enhancement of chlorophyll synthesisin etiolated cells in darkness as well as in light; the ALA-enhancedrate of dark chlorophyll synthesis, however, was much lowerthan the rate in light without added ALA. ALA was replaceablewith succinic acid plus glycine in light, but not in the dark,for enhancement of chlorophyll formation, (iii) Adding glucose,fructose, galactose, glycerol or acetate—at concentrationsmuch lower than those previously shown to induce "bleaching"of green algal cells-caused a more or less marked suppressionof light-induced greening in etiolated cells, (iv) Added glucosealmost instantaneously and completely stopped chlorophyll synthesisin light as well as in darkness either with or without addedALA. On the basis of these and other results, a tentative schemeis presented for the enhancing effects of light and the suppressiveeffects of glucose on chlorophyll formation in algal cells. (Received April 1, 1970; )     

Time Dependence of Phytochrome-mediated Carotenoid and Chlorophyll Synthesis in Sorghum bicolor L

In 5-day-old etiolated Sorghum seedlings, red light irradiationfor 1 s enhanced carotenoid and chlorophyll accumulation, and5 min of red light treatment saturated the photoresponse. Thedegree of red/far-red photoreversibility of carotenoid accumulationwas dependent on the age of the plant. No significant escapefrom far-red reversibility was observed up to 30 min after theonset of a saturating red light pulse in 5-day-old etiolatedseedlings. Thereafter, the escape was relatively fast and completedwithin 180 min. Sorghum bicolor L, carotenogenesis, phytochrome, time dependence, chlorophyll synthesis     

Effects of Intermittent and Continuous Light on the Chlorophyll Formation in Etiolated Plants at Various Ages

The effect of the tissue age of dark-grown bean plants on the chlorphyll formation under continuous illumination or short impulses of white light has been studied. It was found that the protochlorophyllide present in the tissue is age-dependent and reaches a plateau at about 10 days of age, as judged by the chlorophyll formed in etiolated plants of various ages after 5 min illumination. The amount of chlorophyll a and chlorophyll b formed under short impulses of while light increases up to about 9 days of age and thereafter decreases. However, the decrease in chlorophyll a is sharper than that of chlorophyll b, the amount of which remains almost constant. The ratio of chlorophyll a lo chlorophyll b under the short impulses of white light is higher in the younger plant. Similar results are obtained after transfer of the plants from the flashing light to continuous illumination In the young plant there is no lag phase in the chlorophyll biosynthesis while as the age is increased the lag phase is evident and its duration increases as the plant ages. After protochlorophyllide phototransformation under continuous illumination the lag phase in chlorophyll biosynthesis is also age-dependent. Leaves up to 5 days old show no lag phase in chlorophyll synthesis; after this point, however, the lag phase's duration increases continuously with age.     

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.     

Porphyrin metabolism in chill-stressed seedlings of Scots pine (Pinus sylvestris)

Scots pine ( Pinus sylvestris L.) is generally resistant to chilling temperatures. Porphyrin metabolism under low temperature stress was studied in etiolated seedlings of Scots pine. Low temperatures affect porphyrin accumulation in at least 3 different temperature sensitive sites: 1) the light activated accumulation of 5-aminolevulinic acid, a porphyrin precursor, 2) the metabolism of 5-aminolevulinic acid to form porphyrins and 3) a preferential accumulation of chlorophyll a over chlorophyll b . The temperature sensitivity of pine is compared to maize ( Zea mays L.), a chilling sensitive plant.     

Light-regulated pigment interconversion in pheophytin/chlorophyll-containing complexes formed during plant leaves greening

Upon illumination of etiolated maize leaves the photoconversion of protochlorophyllide Pchlide 655/650 into chlorophyllide Chlide 684/676 was observed. It was shown that chlorophyllide Chlide 684/676 in the dark is transformed into pheophytin Pheo 679/675 and chlorophyll Chl 671/668 by means of two parallel reactions, occurring at room temperature: Chlide 684/676. The formed pheophytin Pheo 679/675 was unstable and in the dark was transformed into chlorophyll Chl 671/668 in a few seconds: Pheo 679/675 Chl 671/668. The last reaction is reversed by the light: Chl/668 Pheo 679/675. Thus, on the whole in the greening etiolated leaves this process occurs according to the following scheme:The observed light-regulated interconversion of Mg-containing and Mg-free chlorophyll analogs is activated by ATP and inhibited by AMP.Abbreviations Chl- chlorophyll - Chlide- chlorophyllide - Pchlide- protochlorophyllide - Pheo- pheophytin - PS II RC- Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to the maximum position of the low-temperature fluorescence band (nm), the second number to the maximum position of the longwave absorption band     

Role of Light in the Appearance of Glutamine Synthetase in Leaves of Pennisetum glaucum

Leaves of Pennisetum [Pennisetum glaucum (L) HHB 67] seedlings contained two isozymes of glutamine synthetase (GS, EC 6.3.1.2): cytosolic GS1 and chloroplastic GS2. Leaves of seedlings grown in light for seven days contained about twofold higher GS activity than etiolated leaves. In both light and dark grown seedlings, total GS, GS1 and GS2 activity declined with plant age with more pronounced effect in leaves of etiolated seedlings, and GS2 declined at a much faster rate than GS1. Exposure of etiolated seedlings to light markedly enhanced GS1 and GS2 activity. This increase in activity was not affected by cycloheximide, precluding light dependent de novo synthesis of the enzyme. Treatment of etiolated seedlings with photosynthetic inhibitor, dichlorophenyl dimethyl urea (DCMU) inhibited light dependent appearance of GS. Exogenous supply of sucrose to dark grown seedlings greatly increased the GS activity in dark. These results suggest that light-mediated stimulation in activity of GS in Pennisetum leaves is dependent on photosynthetic reaction.     

THE EFFECTIVENESS OF THE SPECTRUM IN CHLOROPHYLL FORMATION

1. Although the carotenoid pigments are present in large concentration in the plastids of etiolated Avena seedlings as compared with protochlorophyll, the pigment precursor of chlorophyll, it is possible to show that the carotenoids do not act as filters of the light incident on the plant in the blue region of the spectrum where they absorb heavily. This suggests that the carotenoids are located behind the protochlorophyll molecules in the plastids. 2. Since the carotenoids do not screen and light is necessary for chlorophyll formation, an effectiveness spectrum of protochlorophyll can be obtained which is the reciprocal of the light energy necessary to produce a constant amount of chlorophyll with different wavelengths. The relative effectiveness of sixteen spectral regions in forming chlorophyll was determined. 3. From the effectiveness spectrum, one can conclude that protochlorophyll is a blue-green pigment with major peaks of absorption at 445 mµ, and 645 mµ, and with smaller peaks at 575 and 545 mµ. The blue peak is sharp, narrow, and high, the red peak being broader and shorter. This differs from previous findings where the use of rougher methods indicated that red light was more effective than blue and did not give the position of the peaks of absorption or their relative heights. 4. The protochlorophyll curve is similar to but not identical with chlorophyll. The ratio of the peaks of absorption in the blue as compared to the red is very similar to chlorophyll a, but the position of the peaks resembles chlorophyll b. 5. There is an excellent correspondence between the absorption properties of this "active" protochlorophyll and what is known of the absorption of a chemically known pigment studied in impure extracts of seed coats of the Cucurbitaceae. Conclusive proof of the identity of the two substances awaits chemical purification, but the evidence here favors the view that the pumpkin seed substance, which is chemically chlorophyll a minus two hydrogens, is identical with the precursor of chlorophyll formation found in etiolated plants.     

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.

     

Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons

We conducted a series of experiments to assess the effects of oxidative stress on chlorophyll biosynthesis in the vascular plant Cucumis sativus (cucumber). Specifically, cucumber cotyledons were treated with 100 μ M methyl viologen (MV) and subsequently exposed to dark (0 μE m⁻² s⁻¹), low light (40–45 μE m⁻² s⁻¹), or high light (1500–1600 μE m⁻² s⁻¹). Following treatment, extracts of these samples were subjected to high-performance liquid chromatography (HPLC) to quantitate the accumulation of chlorophyll biosynthetic pathway intermediates. The results of these analyses revealed significant accumulation of Mg-protoporphyrin IX monomethyl ester (Mg-proto IX ME) in green (14-h illuminated) as well as in etiolated cotyledons with MV treatment. These data suggest that MV-induced oxidative stress may have inhibited Mg-proto IX ME cyclase activity. Upon exposure to high light, in the presence or absence of MV, both green and etiolated cotyledons predominantly accumulated protoporphyrin IX (Proto IX). These elevated levels of Proto IX might be attributable to attenuated activity of any or all of the following enzymes: Mg-chelatase, Fe-chelatase and protoporphyrinogen IX oxidase. We also observed that MV-induced oxidative stress impacts on chlorophyll biosynthesis to a greater extent than on photosystem II. These results demonstrate that oxidative stress impedes key steps in chlorophyll biosynthesis by either directly or indirectly inhibiting the activity of these enzymes.     

The effect of kinetin on chlorophyll synthesis in ageing etiolated barley leaves exposed to light

Etiolated barley seedlings lose the ability to produce chlorophyll and soluble protein on exposure to light with increasing age. Similarly, the production of δ-aminolaevulinic acid-dehydratase and succinyl-CoA synthetase is decreased in older etiolated leaves exposed to light. The rate of protochlorophyllide₆₅₂ regeneration decreased well before the rates of exogenous δ-aminolaevulinic acid conversion to protochlorophyllide₆₃₂ was affected by ageing. Application of kinetin retarded these ageing symptoms in the etiolated leaves.     

Effect of phytochrome activation followed by prolonged dark incubation on chloroplast development in etiolated cucumber cotyledons

The inhibitory effect of a short red light pulse followed by prolonged dark incubation on chlorophyll accumulation in etiolated cucumber cotyledons ( Cucumis sativus L. cv. Elem) was reflected in the development of the internal membrane system of the mesophyll plastids. Dark incubation for 24 h after phytochrome activation produced the characteristic accelerating effect OB chlorophyll synthesis and chloroplast development. However, longer intervening dark periods (48, 72 and 96 h) before white light exposure resulted not only in a diminished capacity to concentrate chlorophyll, but also in an impaired ability to form grana. The absence of stacking was consistent with a high chlorophyll a to chlorophyll b ratio.     

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.     

Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.     

Effects of light quality on the chloroplastic ultrastructure and photosynthetic characteristics of cucumber seedlings

To understand how light quality influences plant photosynthesis, we investigated chloroplastic ultrastructure, chlorophyll fluorescence and photosynthetic parameters, Rubisco and chlorophyll content and photosynthesis-related genes expression in cucumber seedlings exposed to different light qualities: white, red, blue, yellow and green lights with the same photosynthetic photon flux density of 100 μmol m^?2 s^?1. The results revealed that plant growth, CO₂ assimilation rate and chlorophyll content were significantly reduced in the seedlings grown under red, blue, yellow and green lights as compared with those grown under white light, but each monochromatic light played its special role in regulating plant morphogenesis and photosynthesis. Seedling leaves were thickened and slightly curled; Rubisco biosynthesis, expression of the rca, rbcS and rbcL, the maximal photochemical efficiency of PSII (Fv/Fm) and quantum yield of PSII electron transport (Ф_PSII) were all increased in seedlings grown under blue light as compared with those grown under white light. Furthermore, the photosynthetic rate of seedlings grown under blue light was significantly increased, and leaf number and chlorophyll content of seedlings grown under red light were increased as compared with those exposed to other monochromatic lights. On the contrary, the seedlings grown under yellow and green lights were dwarf with the new leaves etiolated. Moreover, photosynthesis, Rubisco biosynthesis and relative gene expression were greatly decreased in seedlings grown under yellow and green light, but chloroplast structural features were less influenced. Interestingly, the Fv/Fm, Ф_PSII value and chlorophyll content of the seedlings grown under green light were much higher than those grown under yellow light.     

Phytochrome Effects on the Relationship between Chlorophyll and Steady-State Levels of Thylakoid Polypeptides in Light-Grown Tobacco

The effects of phytochrome status on chlorophyll content and on steady-state levels of thylakoid proteins were investigated in green leaves of Nicotiana tabacum L. plants grown under white light. Far-red light given either as a pulse at the end of each photoperiod, or as a supplement to white light during the photoperiod, reduced chlorophyll content per unit area and per unit dry weight. These differences were also observed after resolving chlorophyll-containing polypeptides by gel electrophoresis. Chlorophyll a:b ratio was unchanged. Both Coomassie blue-stained gels and immunochemical analyses showed that, in contrast to the observations in etiolated barley (K Apel, K Kloppstech [1980] Planta 150: 426-430) and pea (J Bennett [1981] Eur J Biochem 118: 61-70) seedlings, and in etiolated tobacco leaves (this report), in fully deetiolated tobacco plants changes in chlorophyll content were not correlated with obvious changes in the steady-state levels of thylakoid proteins (e.g. light-harvesting, chlorophyll a/b-binding proteins).     

Calcium and calcium receptor CAS promote Arabidopsis thaliana de-etiolation

As a second messenger, the free cytosolic calcium ion (Ca(2+)) plays important roles in many biochemical and physiological processes including photosynthesis in plants. In this study, we investigated morphological changes, chlorophyll accumulation and chloroplast development during early photomorphogenesis in etiolated seedlings of both Arabidopsis thaliana wild type (WT) and those with the antisense of CAS, a calcium sensor (CASas). Seedlings were grown at high, medium and low Ca(2+) concentrations to identify the roles of Ca(2+) and CAS in de-etiolation and chloroplast development. The results demonstrated that Ca(2+) and CAS are correlated with de-etiolation of A. thaliana after light exposure. High Ca(2+) significantly increased chlorophyll content and improved chloroplast development in both A. thaliana WT and CASas etiolated seedlings during de-etiolation. The analysis by western blot and real-time fluorescent quantitative polymerase chain reaction indicated that the expression levels of CAS mRNA and protein were upregulated by white light and external Ca(2+) significantly. Etiolated CASas plants showed much lower chlorophyll content and delay of chloroplast development as compared with WT plants, indicating that CAS functions in de-etiolation. All together, we concluded that the de-etiolation in A. thaliana was promoted by the high Ca(2+) concentration and CAS expression to a certain extent.