Stages Of Greening Research Articles

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem I CP complexes were observed before the photosystem II and light-harvesting CP complexes during the initial stages of barley chloroplast development. Photosystem I activity was observed before the photosystem I CP complex was detected whereas photosystem II activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem I remained constant whereas the amount of chlorophyll associated with photosystem II and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development.

On the Correlation between Oxygen Uptake in Plastids of Greening Etiolated Oat Leaves and Pigment Photooxidation

Abstract Changes in oxygen evolution or uptake in illuminated plastids were studied during the early stages of greening in etiolated leaves. An oxygen uptake was observed in etioplasts, which persisted even after the appearance of oxygen evolution. This oxygen uptake was most pronounced in etioplasts or in plastids prepared after redarkening the plants. Incubation of pre-illuminated etioplasts in the presence of NADPH resulted in an inhibition of the uptake in a subsequent illumination. Addition of NADPH had three further consequences: a shift of the chlorophyllide absorbance band to 682 nm, an inhibition of pigment photodestruction and the appearance of a light-induced fluorescence transient at room temperature. Inhibition of pigment photodestruction by NADPH was maximal when the experimental conditions favoured the formation of the P682 chlorophyllide-protein. It is inferred that the oxygen uptake seen under these conditions is due to pigment photooxidation. Inhibition of both phenomena by NADPH is ascribed to the specific interaction of the nucleotide with the pigment-protein complexes and to the ability of these complexes to undergo the light-induced chlorophyllide microcycle, described by Franck and Inoue (Photobiochem. Photobiophys. 8, 85-96, 1984). In the leaf, NADPH is thought to play the same role.

Oxygenic photoreduction of methyl viologen and nicotinamide adenine dinucleotide phosphate without the involvement of photosystem I during plastid development

Studies on the appearance of various electron transport functions were followed during greening of etiolated cucumber cotyledons. Appearance of dichlorodimethoxy-p-benzoquinone, dimethyl quinone, tetramethyl-p-phenylenediamine, dichlorophenol indophenol and ferricyanide Hill reactions were observed after 8h of greening. However, photoreduction of methyl viologen (MV) and nicotinamide adenine dinucleotide phosphate (NADP) was observed from 2h of greening. Variable fluorescence, which is a direct indication of water-splitting function, was observed from 2h of greening in cotyledons, thylakoid membranes and photosystem II (PSII) particles. The decrease in variable fluorescence in the presence of MV (due to rapid reoxidation of Q −) observed from early stages of greening confirmed the photoreduction of MV by PSII. The early development of water-splitting function was further confirmed by the abolition of variable fluorescence in thylakoid membranes and PSII particles by heat treatment and concomittant loss of light dependent oxygen uptake in the presence of MV in heat treated chloroplasts. However, the photoreduction of MV and NADP was insensitive to intersystem electron transport inhibitors, dichlorophenyl dimethylurea or dibromomethyl isopropyl-p-benzoquinone till 8h of greening. Though the oxidation of intersystem electron carrier cytochrome f was observed from early stages of greening, the reduction of cytochrome f was not observed till 8h of greening. All these observations confirm that during early stages of greening MV and NADP are photoreduced by PSII without the involvement of intersystem electron carriers or the collaboration of PSI. Since these observations are at variance with the currently prevalent concept (Z-Scheme) of the photosynthetic generation of reducing power, which requires definite collaboration of the two photosystems, an alternate electron flow pathway is proposed.

Oxygen‐quenched chlorophyll a fluorescence and electron transport in barley during greening

Seedlings of barley (Hordeum vulgare L. cv. Conquest) were dark‐grown for 7 days and then transferred to light. The time courses of chlorophyll a fluorescence induction underwent changes during greening periods of from 3 to 48 h. Yields of variable fluorescence during greening correlated with electron transport capacity via photosystem II (PS II) except at the early stage from 3 to 6 h. The discrepancy may result from there being only a small amount of light harvesting complex associated with PSII for 3 to 6 h greening. Oxygen quenching effects were interpreted as indicating the development of the electron transport system and the organization of light harvesting complex associated with PSII. The most intensive O2 quenching of relative fluorescence is found during the early stage of greening when the ratio of the primary quinone electron acceptor (Q) to chlorophyll is high.

The Effects of δ-Aminolevulinic Acid and Inhibitors of RNA and Protein Synthesis on Phytochrome Mediated Chlorophyll Accumulation inSorghum bicolor

In 5 - d - old etiolated seedlings of Sorghum bicolor, 12 h of darkness after 5 min in red light eliminated a lag before the accumulation of chlorophylls in subsequent continuous white light. Increasing the dark period to 24 h and 36 h, increased the rate of chlorophyll accumulation in the later stages of greening. Exogenous δ - aminolevulinic acid neither completely removed the lag, nor increased the rate of chlorophyll accumulation. Cycloheximide (25 µg ml -1 ) and 6 - methyl purine (5.0 µg ml -1 ), given continuously or only until the 12 h dark period following the red light irradiation, restored the lag and decreased the rate of chlorophyll accumulation. D - threo - chloramphenicol (400 µg ml -1 ) also decreased the rate of chlorophyll accumulation but did not restore the lag. Addition of these inhibitors even 12 h after red light irradiation decreased the rate of chlorophyll accumulation. Rifampicin (Rifamycin SV, 400 µg ml -1 ) did not have such effects.

Light-Induced Accumulation and Stability of Chlorophylls and Chlorophyll-Proteins during Chloroplast Development in Radish Seedlings

Abstract Illumination of 3 day old etiolated radish seedlings with continuous white light results in a progressive accumulation of chlorophyll a and b. Both pigments are bound in a different way to the thylakoid chlorophyll-proteins, which appear parallel to the formation of chlorophylls. By applying the SDS-PAGE method to SDS-digested chloroplasts, it was possible to show that the chloroplasts of radish cotyledons contain the typical chlorophyll proteins LHCP1-3, CPa, CPI and CPIa which have been found in other plants. Between LHCP1 and CPI an additional chlorophyll protein is detected with the spectral properties of a LHCP; it is termed here LHCPy. When the green plants are transferred to continuous darkness, chlorophylls and the chlorophyll-proteins are progressively degraded. At an early stage of greening chlorophyll b is destroyed at a much higher rate in darkness than chlorophyll a, which yields high chlorophyll a/b ratios. This is paralleled by a faster decrease in the level of the corresponding chloro­phyll a/b-protein LHCP3 than of CPI. At a later stage of greening, after the end of the logarithmic chlorophyll accumulation, the chlorophylls a and b and also the LHCP3 and CPI are destroyed in continuous darkness at equal rates; the a/b ratios and the LHCP3/CPI ratios are then little different from the light control. The data indicate that at an early stage of greening the light-harvesting chlorophyll a/b-protein LHCP3 is less stable than the other chlorophyll-proteins (CPI, CPIa, CPa), which contain pre­ dominantly chlorophyll a. The ratio chlorophyll a to β-carotene (a/c ratio) of CPIa, CPI and CPa is about 10, while that of the LHCP1-3 is found to be between 150 to 300. We therefore propose using the a/c ratio to define the chlorophyll-proteins which, besides the absorption spectra, is the most suitable parameter.

Genetic control of chlorophyll biosynthesis by the plastome in some Oenothera species (subgenus Munzia)

Reciprocal differences in the rates of chlorophyll (Chl) formation during early stages of greening are observed in hybrid seedlings with identical genomes derived from reciprocal crosses between Oenothera berteriana (=villaricae) and Oe. odorata (=picensis), subgenus Munzia. In the presence of levulinic acid (LA), a competitive inhibitor of 5-aminolevulinic acid (ALA) dehydratase, ALA accumulated in the cotyledons and chlorophyll production was reduced in a stoichometric ratio. Accumulation of both Chl in untreated tissue and of ALA in seedlings incubated with LA is much more rapid in cotyledons with berteriana plastids than in those with odorata plastids. No difference was found between the inhibitor constants for LA of ALA dehydratase extracted from seedlings with either berteriana or odorata plastids. ALA formation is not limited by the availability of possible precursors. ALA dehydratase and the porphobilinogenase complex (PBGase) are present in abundance and in equal amounts in cotyledons with either berteriana or odorata plastids. It is concluded that the different capacities of the ALA synthesizing system fully account for the different rates of Chl formation in the seedlings with identical genomes and different plastid types.

Light and the Correlation of Chloroplast Development and Coupling of Phosphorylation to Electron Transport

Coupling of phosphorylation to electron transport was examined by measuring the photosynthetic control ratio for broken wheat plastids isolated from seedlings at different greening stages. The photosynthetic control ratio progressively increased during greening and tight coupling was noted after granal stacking and thylakoid elongation. ADP impaired nonphosphorylating (state 2) electron transport rates of plastids at extremely early stages of greening and interfered with photosynthetic control measurements. Partially developed plastids exhibited low nonphosphorylating electron flow rates but did not exhibit high phosphorylating or uncoupled electron transport rates to the same extent as nearly developed plastids. Prolamellar body dispersal, primary thylakoid production, and the development of photosynthetic control were stimulated equally by 48 minutes of low irradiance, in cycles of 2 minutes every 2 hours, or by 9 hours of continuous light of moderate irradiance. Wheat plastids that greened for 6 hours in continuous light of moderate intensity did not exhibit photosynthetic control or much differentiation beyond the etioplast stage. It is concluded that plastid differentiation and the development of photosynthetic control early in greening under continuous light were limited by developmental time (dark time) rather than by either light intensity or duration.

Transcription products associated with developing pea chloroplast membranes

AbstractRNA fractions from leaves of pea (Pisum sativum L.) were labelled by placing dark‐grown shoots in 32P‐phosphate for 1 h in darkness and 3 h in light. Membrane‐associated RNA and soluble RNA was isolated from an organelle fraction containing plastids and mitochondria. A cytoplasmic RNA fraction was also isolated. These RNA fractions were hybridized to nuclear, chloroplast or mitochondrial DNA isolated from fully green leaves. The organelle RNA fractions hybridized significantly only to chloroplast DNA. This result suggests that at early stages of greening, light initiates transcription of part of the chloroplast DNA. Some of the newly transcribed RNA species are associated with the developing plastid membranes.

Translocation and Phosphorylation of Adenine Nucleotides by Mitochondria and Plastids During Greening

Summary Isolated plastids from greening etiolated Avena laminae only showed significant rates of ascorbate/diaminodurene-linked photophosphorylation after 4h of illumination. Mitochondria from similar tissues by contrast exhibited a pronounced increase in respiratory chain phosphorylation driven by externally supplied NADH using a rotenone-insensitive electron flow pathway specific to certain plant mitochondria. An investigation of adenine nucleotide transport across the envelope membranes of the two types of organelles demonstrated substrate saturation with respect to ATP and ADP at all stages of development. The velocities of transport for ADP and ATP showed optima for plastids and mitochondria at 3h and 1h of illumination respectively. The adenine nucleotide translocator of mitochondria differed from that of plastids in exhibiting different affinities between the two nucleotides (Km ADP = 27 μM, Km ATP = 100 μM). Both organelles showed a dependency of the rate of adenine exchange upon the stage of greening and preferred to release ATP or ADP in exchange for ATP, ADP and in the case of mitochondria also AMP; whilst GTP, UTP and CTP showed much lower or negligible rates of exchange. The adenine translocator systems of both plant mitochondria and plastids proved to be relatively insensitive to carboxyatractyloside exhibiting only 50% and 30% inhibitions respectively with concentrations as high as 1 mM. The results are discussed with respect to energy supply for chloroplast development.

Intensity and Decay Half-Time of the Electron Paramagnetic Resonance Signal I in Bean Chloroplasts at Various Stages of Greening

The size of EPR signal I and its decay half-life were measured on chloroplasts isolated from primary bean leaves after illumination of 7 d old dark-grown seedlings. The results indicate that the ratio of P700/total chlorophyll decreased approximately 2-fold only between the 6th and 24th hour of illumination. The relative size of the signal increased with increasing light intensity up to 360 W m~2 in all stages of greening studied. Addition of HgCl2 (1 mM) caused an increase in the magnitude of the signal, especially at lower light intensities. Treat ment of isolated chloroplasts with DCMU (20 //M) did not have any effect on the intensity of the signal at the early stages of greening, that is 6 or 12 h of illumination, but it caused a slight enhance ment after 24 or 48 h of illumination. The higher intensity used for the signal production the smaller the enhancement observed. The addition of HgCl2 caused an increase in decay half-life of the signal 20-fold or 35-fold after 6 h or 48 h of greening, respectively. In DCMU-treated chloroplasts, an enhancing effect becomes visible after 12 h of greening and subsequently it gradually increased. The data obtained were interpreted in terms of an alteration in efficiency of the re-reduction of P700+ under different conditions and different stages of greening.

Kinetics of mitochondrial phosphate transport and rates of respiration and phosphorylation during greening of etiolated Avena leaves

Using the technique of silicone oil filtration of organelles and the inhibitor stop method, the kinetics of transport of inorganic phosphate across the inner mitochondrial membrane were tested in relation to different stages of greening (0 to 24 h) of etiolated laminae of Avena sativa L., and compared to the rates of oxygen consumption and ATP formation. The results demonstrate that there is a pronounced increase in phosphate transport after 3 h of greening, reaching values for Vmax (about 17 μmol mg protein(-1) h(-1)) that are three times as high as those measured with mitochondria from etiolated tissue. This is also mirrored by the rates of respiration and oxidative phosphorylation. After 24 h of light treatment (4 Klx), respiration and ATP formation, as well as V decreased again to levels below those of the etiolated stage. In contrast to V, there was no change in the affinity between inorganic phosphate and the binding sites of the transporting systems involved, as indicated by a rather constant Km (0.23 mM) for phosphate transport. Of the inhibitors of phosphate transport tested, mersalyl and methyl mercuric iodide were most efficient with identical characteristics of inhibition; but compared to animal mitochondria, the concentrations needed to result in similar amounts of inhibition, were more than ten times higher. The results are discussed with respect to plastid development.

Chloroplast Biogenesis

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.

Development of the cation-induced stacking capacity during the biogenesis of higher plant thylakoids

We studied the capacity of the thylakoid membrane to form grana stacks in the presence of cations, monovalent or divalent, added to N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine “low-salt” disorganized plastids during their greening. Grana stacking was monitored by the yield of heavy subchloroplast fractions separated by differential centrifugation after digitonin disruption of plastids (J. H. Argyroudi-Akoyunoglou, 1976, Arch. Biochem. Biophys., 176, 267–274) . Primary thylakoids of the agranal protochloroplasts formed in periodic light do not show the cation-induced stacking capacity of the mature green chloroplast thylakoids. Similarly, the cation effect saturates at lower cation concentrations in mature chloroplasts than in plastids of the early stages of greening. The capacity for cation-induced stacking and for saturation of the effect at low cation concentrations appears gradually after exposure to continuous light and parallel to the appearance of chlorophyll b and the polypeptides of the 25,000–30,000 molecular weight range of lipid-free thylakoids, probably derived from the chlorophyll b-rich chlorophyll protein Complex II. The thylakoid peripheral stroma proteins ribulosediphosphate carboxylase and the coupling factor protein are not involved in the cation-induced stacking, since their removal (H. Strotmann, H. Hesse, and K. Edelmann, 1973, Biochim. Biophys. Acta, 314, 202–210) does not affect the thylakoid aggregation.

Changes in the polypeptide composition of internal membranes of barley plastids during greening

A method is described for obtaining good yields of purified internal membranes of plastids from greening barley seedlings. The procedure is suitable, without modification, for isolating intact plastids from etiolated seedlings and all early stages of greening. Electron microscopy was used to identify the types of contaminants present in preparations obtained by different methods, and to assess the degree of contamination in purified membranes. Gel electrophoresis of the sodium dodecyl sulphate-solubilised membrane polypeptides established that the proteins pelleted during flotation were a major component of unpurified membrane preparations from etioplasts and plastids greened for 9 hours or less. The polypeptide composition of purified etioplast membranes consisted of 35 bands. At least 15 of these disappeared during subsequent greening. After only 3 hours of illumination, the internal membranes contained almost all of the 43 polypeptides present in mature chloroplast thylakoids. The changes in polypeptide composition from 3–24h were mainly quantitative in nature.

Comparative Effects of Several Inhibitors of Chloroplast Thylakoid Membrane Synthesis in Greening Jack Bean

The in vivo effects of several inhibitors of protein and RNA synthesis have been compared with regard to chlorophyll and RNA synthesis and ultrastructural development of greening jack bean (Canavalia ensiformis [L.] DC.) leaves. Inhibitors of ribosome function (chloramphenicol, aminotriazole, streptomycin, and kanamycin) reduced chlorophyll synthesis 40%-70%, inhibited RNA synthesis by 30%-40%, and arrested chloroplast development at similar stages of greening. Cycloheximide had identical effects on chlorophyll synthesis and chloroplast development but no effect on RNA synthesis. Rifampicin, an inhibitor of procaryotic RNA polymerases, had no effect on chloroplast development. It is possible that inhibitors of chloroplast membrane synthesis may act by altering energy metabolism as well as by affecting RNA and protein synthesis.

Mn content changes in wheat etioplast membranes during greening under intermittent or continuous light

Study of Mn content changes in wheat etioplasts during greening under intermittent or continuous light reveal an heterogeneity in the development of the chloroplast Mn pool. Under intermittent light plastids contain only the Tris extraction-resistant Mn pool. Such plastids are known to have no O 2 evolution capacity. The formation of the tightly bound Mn pool appears not to be a light-requiring process. But under intermittent light followed by 15 min continuous light plastids contain, like the control chloroplasts, two distinct Mn pools, the Tris extraction-resistant and the Tris-extracted pools. Such plastids are known to be capable of evolving O 2 in the presence of Hill reagents. The formation of the loosely bound Mn pool is a light-requiring process. Under continuous light, only the loosely bound Mn pool increases rapidly in the early stages of greening. These results suggest that protein complexes capable of Mn binding pre-exist in etioplasts and Mn atoms are light-inserted into the membrane complexes a as chlorophylls are synthesized.

Growth Responses of Rice Seedlings to Light Wavelengths : II. Effect of light wavelength on chlorosis of seedlings

This study deals with the effect of various light wavelengths on chlorosis of the leaves of rice seedlings treated with darkness in the promoting germination state and greened under high solar radiation of 529 and 550 cal·cm-2·day-1. 1. Under condition of 529 cal·cm-2·day-1 solar radiation in the greening stage, blue and green film inhibited chlorosis, while red and purple had no effect. 2. With higher transmission rate in the visible region and with lower B/R ratio of light, the degree of chlorosis was increased, but the chlorophyll content was decreased. 3. With high solar raditttion of 550 cal·cm-2·day-1, a significant positive correlation between the degree of chlorosis and the transmission rate of light in the 600-700 nm region was observed. 4. The transmission rates through a single layer of etiolated 1st leaf and of greened 2nd leaf sheath were 83% and 140%, respectively. The spectral transmission of both the etiolated 1st leaf of 600-700 nm light and the greened 2nd leaf sheath of 500-600 nm light were relatively high. The decrease of transmission rates rather than the changes of spectral transmissions in leaves seemed to contribute to the inhibition of chlorosis.

Fluxes of gibberellic and abscisic acids, together with that of adenosine 3?,5?-cyclic phosphate, across plastid envelopes during development

Application of the technique of silicone oil centrifugation to etioplasts and etio-chloroplasts isolated from greening etiolated Avena sativa L. laminae has shown that etioplast envelope membranes are relatively impermeable to gibberellic acid, abscisic acid and adenosine 3',5'-cyclic phosphate. However, there is a marked increase in permeability for all in the early stages of greening, particularly after 2 h. All fluxes are reduced thereafter but a high level of abscisic acid still remains.

Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations.

1. Chlorophyll (ide) formation from protochlorophyll (ide) that is normally inactive was demonstrated in etioplast membranes isolated from maize and barlley plants, the process being dependent on intermittent illumination and the addition of NADPH. 2. The addition of NADPH to the membranes was shown to result in the conversion of inactive protochlorophyll (ide) absorbing at about 630 nm into a form(s) with light-absorption maxima at about 640 and 652 nm, both of which disappear when chlorophyll (ide) is formed on illumination. 3. The temperature-dependence of the activation process and its response to a variety of reagents were examined. From these, the conclusion is drawn that -SH groups are involved in the activation but in the active complex these are unavailable for reaction with -SH reagents. 4. Evidence is presented for the occurrence of glucose 6-phosphate dehydrogenase activity within etioplasts and the suggestion is made that the oxidative pentose phosphate pathway can provide the NADPH required for chlorophyll biosynthesis during the early stages of greening.