Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Photoregulation

The preparation of a rabbit antibody to ribulose-1,5-bisphosphate carboxylase (RuBPCase) from Euglena gracilis and its use to quantitate RuBPCase in dark- and light-grown cells and during light-induced chloroplast development (greening) are described. Light-grown Euglena have at least 36 times more RuBPCase than dark-grown Euglena. Light is required for both the initiation and continued increase in net synthesis of RuBPCase over the dark level: brief illumination 12 hours before exposure to continuous light eliminates the lags in the accumulation and increase in activity of RuBPCase (as well as in chlorophyll accumulation); net synthesis is blocked in greening cells returned to the dark or exposed to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Streptomycin or cycloheximide prevents RuBPCase accumulation when added at the beginning of greening but only partially blocks accumulation when added after 25 hours of greening. After 24 hours of greening, the activity of RuBPCase per milligram chlorophyll continues to increase slowly while concentration of the enzyme per milligram chlorophyll remains constant. This increased activity may be due to activation of the enzyme as well as to net synthesis.     

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)-N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

Change in Levels of Acyl Carrier Proteins in Greening Plants

The levels of acyl carrier proteins (ACP) in greening spinachcotyledons and greening oat leaves were examined by immunoblottingwith antiserum raised against spinach ACP I. Two isoforms ofACP, ACP I and ACP II, were found in spinach cotyledons, asthey were in the green leaves. The level of ACP II was higherthan that of ACP I in etiolated cotyledons. The level of ACPI increased markedly with greening. In the greened cotyledons,the major isoform was ACP I as was the case in green spinachleaves. In oat leaves, two isoforms were also identified, oatACPI (about 12kDa) and ACP II (about 17kDa), which cross-reactedwith the antiserum against spinach ACP I, but which were differentfrom spinach ACPs I and II. The levels of oat ACPs I and IIwere very low in etiolated leaves. The increase in levels ofboth ACPs corresponded to the change in the activity of fattyacid synthesis during illumination for 24 h. During furtherillumination for 24 h, the level of ACP II increased a littlein parallel with the change in the activity of fatty acid synthesis,whereas the level of ACP I increased somewhat more. The functionof oat ACPs I and II is discussed in connection with the formationof chloroplast. (Received March 27, 1989; Accepted September 18, 1989)     

Light induction of phosphoenolpyruvate carboxylase in etiolated maize leaf tissue

An antibody for phosphoenolpyruvate carboxylase was used to isolate and to quantitate the enzyme from greening maize (cv. KOU 6) leaves. The increase in enzyme activity during greening was due to de novo synthesis, which was paralleled by increases in enzyme protein and incorporation of leucine. The light-induced activity was due to one specific isoenzyme. The action spectrum for enzyme synthesis had red and blue peaks.     

Effects of delta-aminolevulinic acid on pigment formation and chlorophyllase activity in French bean leaf

Chloroplast development and chlorophyll biosynthesis are co-regulated. Treatment by levulinic acid resulted in a linear relation in both chlorophyll and carotenoid contents, during greening of etiolated French bean leaf discs. Chlorophyll biosynthesis appeared to control that of caroteins. In the presence of levulinic acid; at different levels, photosystem II (PS II) activity decreased when expressed on a chlorophyll basis. Chlorophyllase activity was increased progressively by increasing levulinic acid concentration. Thus, levulinic acid could be used to arrest the light-induced chloroplast development at a desired phase of greening and acts as determinator of chloroplast development in green tissues.     

Ferredoxin and ferredoxin-NADP-oxidoreductase in leaves ofPhaseolus vulgaris L.

During light-induced greening of 10-dayold etiolated bean seedlings a strong increase is observed of ferredoxin (Fd) and of ferredoxin-NADP-oxidoreductase (FNR; E.C. 1.6.99.4) activity, both known to reside in chloroplasts. The production of Fd starts immediately upon illumination and proceeds almost linearly for at least the next 72 h. It is inhibited by chloramphenicol (CAP) but not by cycloheximide (CHI), beit that in the presence of the latter Fd synthesis was impaired after 48 h of illumination. We conclude that for the elaboration of functional Fd in greening chloroplasts protein synthesis on chloroplast ribosomes is required. The increase of FNR activity shows a lag of about 24 h and is blocked by both CAP and CHI. When CAP is applied at 24 h after the illumination started it has no effect. We suggest that the synthesis of FNR on cytoplasmic ribosomes requires prior synthesis of protein(s) on chloroplast ribosomes.The nature of possible interactions between chloroplasts and cytoplasm is discussed.Abbreviations CAP D-threo-chloramphenicol - CHI cycloheximide - DCIP dichlorophenol-indophenol - DEAE diethylaminoethyl - Fd Ferredoxin - FNR ferredoxin-NADP-oxidoreductase - NAR nitrate reductase - NIR nitrite reductase     

Interaction of Chloroplasts with Inhibitors: Location of Carotenoid Synthesis and Inhibition during Chloroplast Development

The inhibitor SAN 6706 [4-chloro-5-(dimethylamino)-2-(α,α,α,-trifluoro- m-tolyl-3(2H)-pyridazinone] has been used to study the synthesis of carotenes and xanthophylls during the conversion of etioplasts to chloroplasts in developing barley (Hordeum vulgare) shoots. SAN 6706 inhibits carotenoid synthesis and causes an accumulation of phytoene, but it is also a potent inhibitor of chloroplast electron transport. When developing barley is treated with SAN 6706, carotenoid synthesis is inhibited but total photosynthesis is unaffected. The ability of SAN 6706 to inhibit carotenoid synthesis becomes progressively less if etiolated shoots are illuminated for increasing lengths of time before treatment. During the greening of treated barley shoots only light-induced β-carotene synthesis is immediately inhibited; xanthophyll synthesis is not affected until after about 8 hours. The hypothesis that SAN 6706 cannot enter the chloroplast but inhibits carotenoid synthesis from the cytoplasm is discussed, and the question as to whether there are not two separate groups of enzymes for the synthesis of carotenes and xanthophylls is considered.     

Biosynthesis of Ferredoxin-Nitrite Reductase in Rice Seedlings

Changes in ferredoxin-nitrite reductase [EC 1.7.7.1 [EC] ] in etiolatedrice seedlings were followed during induction by nitrate andlight. Etiolated seedlings showed maximal induction of the enzymeactivity during greening with nitrate, while the enzyme activityin etiolated seedlings receiving nitrate in darkness increasedhalf as much as that in nitrate-treated greening plants. Theincrease in nitrite reductase activity during induction coincidedwith an increase in the content of proteins immunoprecipitatedby antibodies raised against spinach nitrite reductase. Lighthad no effect on the induction of the extractable nitrite reductasein the absence of nitrate. Poly(A)⁺-RNA extracted from nitrate-treatedgreening shoots directed the synthesis in a rabbit reticulocyte-lysateof polypeptides immunoprecipitated by spinach nitrite reductaseantibodies. One major polypeptide larger than the native enzymewas found among the translation products, suggesting that nitritereductases in greening rice shoots are synthesized as an precursorform. Analysis of two-dimensional electrophoretograms indicatedthe existence of isoforms of nitrite reductase in rice seedlingswhich had been immunoprecipitated with spinach nitrite reductaseantibodies. ¹To whom all correspondence should be sent. (Received May 15, 1987; Accepted September 7, 1987)     

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30–40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.     

Light-stimulated synthesis of NADP malic enzyme in leaves of maize

Illumination of etiolated maize plants for 80 h brings about a 15-20-fold increase in activity of NADP malic enzyme (EC 1.1.1.40). Increases in NADP malic enzyme protein and in the level of translatable mRNA for this protein occur simultaneously with the activity increase. Radiolabeled amino acids are also incorporated into NADP malic enzyme during this time. These results are consistent with the conclusion that an increase in NADP malic enzyme activity during greening results from de novo synthesis of NADP malic enzyme protein. Polyadenylated RNA extracted from greening maize leaves directs the synthesis in vitro of a protein 12,000 daltons larger than NADP malic enzyme purified from corn leaves. This protein is a precursor of NADP malic enzyme because 1) both the precursor and mature NADP malic enzyme are immunoprecipitated by antibody made against NADP malic enzyme purified from corn leaves, 2) both NADP malic enzyme protein and the level of mRNA for the precursor increase during greening, and 3) peptide maps of the precursor and of mature NADP malic enzyme are very similar. Mature NADP malic enzyme and its precursor (synthesized in vitro) both migrate on sodium dodecyl sulfate-polyacrylamide gradient gels as doublet bands. Peptide analyses show all bands to be structurally related.     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH₃ are discussed.     

Comparative Enzymology of the Glyceraldehyde 3-Phosphate Dehydrogenases from Pisum sativum

Glyceraldehyde 3-phosphate dehydrogenases (EC 1.2.1.12 and 1.2.1.13) have been purified from the seed, root, etiolated, and green shoot of peas (Pisum sativum). These enzymes are tetramers of 140,000 daltons, with subunits of 35,000 daltons. The enzymes differ in isoelectric point. The seed enzyme has a pI of 5.1, and the root enzyme has a pI of 4.5. The cytoplasmic enzyme from etiolated shoots is slightly acidic with a pI of 5.7 to 6.1 and is found in two separable forms. The chloroplast enzyme (from green shoots) is most basic with a pI of 8.0.     

Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS₂) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS₂. The synthesis of GS₂ was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS₂ protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH heavy chain of antibodies - AbL light chain of antibodies - AP acid phosphatase - CH cycloheximide - G6PDH glucose-6-phosphate dehydrogenase - GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - LC lincomycin - NAD-MDH NAD malate dehydrogenase - NADP-G3PDH NADP glyceraldehyde-3-phosphate dehydrogenase     

Sucrose Suppression of Chlorophyll Synthesis in Tissue Culture: Changes in the Activity of the Enzymes of the Chlorophyll Biosynthetic Pathway

Chlorophyll synthesis in carrot root tissue cultures grown ona medium containing sucrose is inhibited. Examination of theactivities of the first enzymes in the chlorophyll biosyntheticpathway shows that the major effect of sucrose upon chlorophyllsynthesis occurs at the stage controlling 5-aminolevulinic acid(ALA) synthesis. The toxic nature of ALA in this tissue precluded its use toalleviate the sucrose inhibition effect but the utilizationof various other precursors for chlorophyll synthesis was consistentwith a block occurring at the level of ALA synthesis. The activities of other enzymes involved in chlorophyll synthesisdecreased in activity paralleling decreases in chlorophyll amount.These latter changes are thought to result as a consequenceof chloroplast degeneration rather than representing a primecause in the loss of greening.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

Studies on S-adenosylmethionine–magnesium protoporphyrin methyltransferase in Euglena gracilis strain Z

1. An enzyme that methylates magnesium protoporphyrin was detected in extracts of light-grown and dark-grown cells of Euglena gracilis. The activity in light-grown cells is two to three times that in cells grown in the dark. 2. The activity is mainly located in the chloroplast fraction from light-grown cells and in proplastids in dark-grown cells. However, in cells grown either in the light or dark, about 15-20% is found in particle-free supernatant. 3. The chloroplast methylating enzyme was solubilized by the action of Tween 80 and partially purified. The properties were investigated. 4. From experiments in which etiolated cells were illuminated in the presence of inhibitors of chloroplast or cytoplasmic protein synthesis, it appears that the methylating enzyme is made on cytoplasmic ribosomes.