Early Stages Of Greening Research Articles

Immunological Studies on Pyruvate Orthophosphate Dikinase in C<sub>3</sub> Plants

Pyruvate orthophosphate dikinase (PPDK) was detected in some C3 plants, wheat, barley, rice and tobacco, by protein blotting using an antibody against maize PPDK, although the amounts were much lesser than those of C4 plants. The PPDK activity in immature grains of rice was specifically immunoprecipitated by the anti-(maize) PPDK antibody. The molecular weight of the subunit of PPDK in all tested C3 plants was similar (ca. 95 kD) to that of maize PPDK, and the fragment patterns of the C3 PPDKs in peptide mapping were also similar to that of maize PPDK. These results suggest that C3 PPDKs have a primary structure similar to that of maize PPDK. In order to obtain information about the expression of PPDK in C3 plants, changes in the enzyme activity and in the amount of PPDK protein were investigated during the greening of rice seedlings. PPDK, which was found in the etiolated seedlings, decreased temporarily in an early stage of greening and then increased. The mechanism of this variation is discussed.

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.

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.

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.

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.

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.

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.

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.

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.

Aspects of structure and photosynthetic competence of Euglena plastids under conditions of greening and degreening.

Dark-grownEuglena cells were exposed to light at both their late stationary phase of growth (“old cells”), and at their late exponential phase (“young cells”). Chlorophyll accumulation was faster and photosynthetic oxygen evolution was detected earlier in the “young” cells. Photosystem I activity was measured at early stages of greening in both “young” and “old” cells. Rate of activity on a chlorophyll basis at these early stages was 6–7 times higher than the constant levels of the late stages. Photosystem II activity developed with illumination and showed a long lag period in “old” cells. In cells returned from light to dark, after seven generations in the dark, photosystem I was seven times more active on a chlorophyll basis than in light-grown cells. Photosystem II activity inceased up to four generations in the dark and decreased thereafter. Chlorophyll was diluted among progeny, whereas oxygen evolution did not show a similar dilution pattern in the dark. Chloroplasts lost their typical structure and were transformed into proplastids. Prolamellar bodies within the transformed plastids were observed after 2–4 generations in the dark. Non-dividing cells, in contrast to the dividing ones, kept their chlorophyll, their ability to evolve oxygen, and their plastidial structure over long periods.

Photosynthesis and apparent proton fluxes in intact cells of greening etiolated barley and maize leaves

Summary Chlorophyll synthesis, formation of thylakoid membranes and of membrane partitions proceeded rapidly after the onset of illumination of 6 days old barley and 7 days old maize seedlings. Consistent with these structural changes photosynthetic O 2 evolution was detectable very early during greening. However, the capacity for photosynthetic CO 2 fixation developed more slowly than O 2 evolution. The interpretation of this unexpected discrepancy between CO 2 fixation and O 2 evolution in the early stages of greening depends largely on whether photosystem I or photosystem II develops earlier during greening. Both cases are discussed. Light dependent apparent H + uptake from the external solution into leaf slices of greening plants develops simultaneously with O 2 evolution rather than with CO 2 fixation. Hence, most of the photosynthesis dependent pH change in the early stages of greening must be independent of CO 2 fixation and HCO 3 − uptake from the solution and more closely related to photosynthetic electron flow.

Quantitative studies on ferredoxin in greening bean leaves.

Two methods of measuring small amounts of the iron-sulphur protein ferredoxin are described. One involves measurements of the signal at g=1.96 produced by reduced ferredoxin in an e.p.r. (electron-paramagnetic-resonance) spectrometer; the other depends on the rate of ferredoxin-dependent electron transport in a chloroplast bioassay measured in an O(2) electrode. These methods of measurement were used to examine the development of ferredoxin during the greening of etiolated bean leaves. Ferredoxin is present in low concentrations in the leaves and cotyledons of 14-day-old etiolated beans (Phaseolus vulgaris L. var. Canadian Wonder), and develops in a linear manner with time when the leaves are illuminated. This synthesis appears to be independent of chlorophyll synthesis during the early stages of greening. However, the chlorophyll/ferredoxin ratio reaches a final value of approx. 360 irrespective of the light intensity, indicating the existence of a control mechanism operative in deciding the stoicheiometry of these components in the mature chloroplast. The ferredoxin synthesis appears to be light-dependent, and red light is the most effective in its promotion. The effect of red illumination is not reversed by far-red light, indicating the absence of a phytochrome control of ferredoxin synthesis. From experiments using specific inhibitors of chloroplast protein synthesis, it is concluded that ferredoxin is synthesized on cytoplasmic ribosomes.

Photosynthetic O2 evolution and apparent H+ uptake by slices of greening barley and maize leaves in aerobic and anaerobic solutions

Apparent rates of photosynthetic O2 evolution and light-dependent pH change (Δ[H+]) were measured in solutions containing leaf slices and bubbled with gas streams of varied composition. Intracellular recycling of O2 and CO2 between photosynthetic and respiratory reaction systems seems to be possible in green cells in the light. As a result of photosynthetic O2 evolution, the tissue and the solution are never strictly anaerobic even after prolonged bubbling with N2. Internal gas exchange is more pronounced in slices from the leaves of maize (a C-4 plant) having the complex Kranz tissue differentiation of mesophyll and bundle sheath, than in the leaves of barley (a C-3 plant).Light-dependent Δ[H+] is observed under all conditions used, i.e. in air, air – CO2, pure N2, and N2 + CO2. During the early stages of greening of etiolated leaves, Δ[H+] is found to be closely related to photosynthetic O2 evolution. After prolonged greening, Δ[H+] is larger in air – CO2 than in air. In pure N2, Δ[H+] is smaller and apparent O2 evolution is larger than in N2 + CO2. These results are discussed in relation to the hypothesis that part of light-dependent Δ[H+] is independent of concomitant photosynthetic CO2 fixation.

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.Plastids showed substantial photochemical oxygen evolution after a seedling greening time of 1 hour. However, a comparison of the relative activity of leaves and plastids at 2 hours and 24 hours of greening suggests that there was some inactivation of greening plastids during isolation. Appreciable photosystem I activity was observed as early as 15 minutes of greening.The synthesis of cytochrome b-559(HP) during greening does not correlate with the onset of oxygen evolution. Cytochrome b-559(HP) was absent from etioplasts and in most preparations of 2-hour plastids. The average amount of cytochrome b-559(HP) at 2 hours of greening was well below the level needed to provide 1 molecule of the carrier for each functional photosynthetic chain. The results suggest that cytochrome b-559(HP) is not essential for oxygen evolution. Cytochrome f, cytochrome b(6), and the low potential form of cytochrome b-559 were present in the etioplast. There was little increase in the levels of these cytochromes during 24 hours of greening.

Light-induced Changes in the Pattern of Protein Synthesis during the Early Stages of Greening of Etiolated Maize Leaves

The effect of light on protein synthesis during the early stages of greening of etiolated maize (Zea mays) leaves was studied using double labeling with leucine and fractionation of proteins by gel filtration and acrylamide gel electrophoresis. The incorporation of labeled leucine into a relatively small number of plastid proteins is effected within the first 30 to 60 minutes of illumination. These proteins do not accumulate with time. When illumination is prolonged, additional proteins are effected.Experiments using inhibitors of protein synthesis suggest that at least some of the proteins effected by 1 hour of illumination might be synthesized in the cytoplasm and not in plastids. Actinomycin D inhibits the incorporation of labeled leucine into some of the protein fractions, but enhances the incorporation into other fractions far above the effect exerted by light.