Stoichiometric analysis of barley plastid ribosomal proteins

We analyzed the protein composition of plastid 70S ribosomes isolated from the stromal fractions of barley plastids by the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR 2D-PAGE). Intactness of the ribosomes was confirmed by the poly(U)-directed phenylalanine polymerization activity and by the reassociation capacity of the subunits into 70S ribosomes. The small and large ribosomal subunits were composed of 23 and 36 proteins, respectively. In addition, one acidic protein associated with ribosomes in low salt buffer but released in high salt buffer was found. The plastid ribosomes contained relatively larger numbers of acidic proteins than prokaryotic ribosomes. Stoichiometric analysis revealed the presence of several ribosomal proteins in low copy numbers, indicating that the ribosomes of plastids were heterogeneous. We also investigated the protein composition of plastid ribosomes from greening barley leaves and found that it did not change during greening.     

Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.     

The light-induced development of nitrate reductase in etiolated barley shoots: An inhibitory effect of laevulinic acid

Induction of nitrate reductase EC 1.6.6.1 in etiolated barley (Hordeum vulgare L., var. Proctor) required continuous illumination and showed a lag period of about three hours. During the first 16 h of illumination the ratio NADH/NAD and NADPH/NADP, taken as a measure of internal oxidation reduction potential, declined. The inhibitor DCMU applied to whole leaves at concentrations shown to inhibit the reduction of cytochrome f by Photosystem 2 light did not inhibit the induction of nitrate reductase nor did it diminish the ratio of reduced to oxidised puridine nucleotides in the early hours of greening. It was concluded that light driven electron flow was not necessary for nitrate reductase induction. Chloramphenicol gave a slight inhibition of nitrate reductase induction. Laevulinic acid was added to greening barley leaves to inhibit tetrapyrrole pigment biosynthesis and plastid development. It strongly inhibited chlorophyll synthesis and nitrate reductase induction, with relatively little effect upon Photosystem 1 and 2 activities in isolated plastids. The activities of other inducible enzymes and control enzymes were little affected by laevulinic acid. Laevulinic acid also inhibited nitrate reductase induction by added nitrate in fully-greened illuminated plants grown in nitrate-free medium and so is unlikely to be acting through inhibition of plastid development. This inhibitor lowered the level of protohaem in whole leaves and plastids of greening barley and it is postulated that it may diminish the protohaem available for the assembly of a cytochrome b component of nitrate reductase.Abbreviations DCMU 3-(3:4-Dichlorophenyl)-1:1-dimethylurea - LA laevulinic acid     

菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

Light-induced changes in the distribution of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase within different cellular compartments of barley (Hordeum vulgare L.)

Changes in the relative content of NADPH-protochlorophyllide oxidoreductase during the light-induced greening of barley plants were measured both in the total leaf extract as well as in intact and broken plastids. The enzyme protein was identified by its apparent molecular weight and its immunological crossreactivity with an antiserum directed against the NADPH-protochlorophyllide oxidoreductase. The monospecificity of the antiserum was tested by two different criteria: i. The antiserum was purified by affinity chromatography. ii. It was demonstrated that the antiserum crossreacts with only those polypeptides which appear to be enzymatically active. In the fraction of broken plastids isolated from leaves of briefly illuminated barley plants the concentration of the enzyme protein was reduced drastically. Our results indicate that this decrease in enzyme protein content is the consequence of an artificial proteolytic breakdown of the membrane-bound enzyme protein. In intact plastids and in the total leaf extract the concentration of the enzyme protein did not change dramatically during the first 4 to 6 h of illumination. However, when the exposure to continuous white light was extended further the concentration of the enzyme protein in intact plastids began to decline rapidly while in total leaf extracts the concentration remained almost constant for the next 10 h of light. These results indicate that part of the enzyme protein may be localized outside of the plastid compartment.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulfate     

Simultaneous Appearance of C-550 and Cytochrome b559HP (High Potential Form) in Barley Leaves Greened under Intermittent Light

Cytochrome b_559HP has been detected by spectrochemical assays in plastids of barley leaves greened under intermittent light (flashed leaves). The amount of cytochrome b_559HP in these plastids was nearly 10-fold lower than in normal chloroplasts when the results were expressed on plastid number. The amount of cytochrome b_559HP phototransformable at -170°C was similar, on a C-550 basis, in the plastids of flashed and normal green leaves. The appearance of the 2 components was simultaneous during the greening process under intermittent light and it is suggested that it was parallel to the increase of appressed regions in thylakoids. The illumination of flashed leaves under continuous light for 5 minutes allowed the appearance of a normal Photosystem-II activity, but had no effect either on the cytochrome b_559HP content of the plastids or on the photoreactivity of this component at low temperature.     

Changes in Chloroplast Lipids during the Development of Photosynthetic Activity in Barley Etio-Chloroplasts

The content of monogalactosyl diglyceride, digalactosyl diglyceride, sulfoquinovosyl diglyceride and phosphatidyl glycerol of gel-filtrated etio-chloroplasts isolated from greening barley seedlings was determined. The development of photosynthetic electron transport, measured as anthraquinone autooxidation, was simultaneously determined with an oxygen electrode. During the first hour of irradiation of the etiolated seedlings the lipid content of the plastids decreased rapidly. The decrease is interpreted as a chlorophyll sensitized photooxidation of the fatty acids of the diglycerides. With artificial electron donors an oxygen uptake was detected after 10 min of greening. With no donors added, a DCMU sensitive oxygen uptake was detected after 2 h. The level of DCMU inhibition increased as the plastid developed and total inhibition was obtained after 5 h. Between 2 and 6 h of greening the lipid content of the plastids stayed constant. During this greening period there was a correlation between the appearance of a DCMU sensitive electron transport and the accumulation of the trans-3-hexadecenoic acid of phosphatidyl glycerol. The trans-3-hexadecenoic acid was present already in the dark-grown seedlings but an increase in content did not occur until after 3 h. The lipid content increased after 6 h of greening. This increase coincided well in time with the formation of grana. The fatty acid composition of the individual lipids, with the exception of phosphatidyl glycerol, and the monogalactosyl diglyceride to digalactosyl diglyceride ratios did not change fundamentally during the greening.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : II. Formation from Glutamate

Intact plastids from greening maize (Zea mays L.) leaves converted [¹⁴C]glutamate and [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg²⁺, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.

The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

     

Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.     

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.     

Lipid biosynthesis by isolated barley chloroplasts in relation to plastid development

The effect of seedling age and of the time of greening on the incorporation of 1-¹⁴C-acetate into lipids by isolated barley (Hordeum vulgare cultivar Svalöf's Bonus) plastids was examined. The fatty acid synthesizing capacity of plastids isolated from 5-day-old seedlings did not increase markedly from zero to 36 hours of greening nor was a light stimulation of fatty acid synthesis observed. However, an increasing capacity for fatty acid synthesis and an increasing light stimulation of this process with greening were attained by the plastids isolated from 7-, 9-, and 11-day-old seedlings.     

Identity of a Group of 13.5-20 kDa Polypeptides Whose Apparent Abundance Decreases during Plastid Development

The identity of a group of 13.5–20 kDa polypeptides whoseabundance decreases during the greening of intermittent-lightgrown barley seedlings was investigated. During greening therelative abundance of the 13.5–20 kDa polypeptides wasinversely related to that of LHC II, which had led others tosuggest a role of these polypeptides in the assembly of theLHC II and/or chloroplast development. The smallest 13.5 kDapolypeptide was identified as histone H4 by N-terminal sequencingof an internal peptide fragment produced by CNBr cleavage. Theentire group of 13.5–20 kDa polypeptides was thereafterverified to be nuclear histones by their similar mobility onSDS-PAGE to that of barley histones and immunoreactivity toyeast histone anti bodies. Their presence results from contaminationof plastid preparations by nucleosomes. Our results unequivocallysubstantiate earlier suggestions of others that polypeptidesoften found to contaminate immature plastids were of nuclearorigin. Methods to reduce or remove the histone contaminationwithout reduction in yield of thylakoids were developed so thattrue changes in the polypeptide content of thylakoids can bestudied during plastid development. (Received December 19, 1991; Accepted August 15, 1992)     

Measurement of heme efflux and heme content in isolated developing chloroplasts

Hemes destined for cytosolic hemoproteins must originate in one of the cellular compartments which have the capacity for heme synthesis, namely the chloroplast or the mitochondria. Since developing chloroplasts from greening cucumber (Cucumis sativus, cv. Sumter) cotyledons are known to contain complete heme and chlorophyll biosynthetic pathways, they were tested for their capacity export hemes. Picomole quantities of heme were measured by reconstitution of the heme with apo-peroxidase and subsequent determination of peroxidase activity. The assay method was sensitive (as little as 0.7 picomole of heme could be detected in a volume of 100 microliters) and was linear with heme concentration. When intact plastids were incubated with apo-peroxidase, a steady-state rate of efflux between 0.12 and 0.45 picomole heme/minute/milligram plastid protein was measured. The efflux rate was not due to plastid breakage and could be enhanced by incubating with the heme precursor, δ-aminolevulinic acid. Cold acetone extraction removed 47 ± 17 picomoles heme/milligram plastid protein from the total b-type heme pool in the chloroplasts (166 ± 9 picomoles heme/milligram protein, by acid-acetone extraction). The reconstitution technique provided a similar estimate of readily exchangeable heme in the plastid, 37 ± 8 picomoles heme/milligram protein (or 6 micromolar in the plastids). These values may be indicative of a `free heme pool' which exists in the chloroplast.     

ATP requirement for mg chelatase in developing chloroplasts

The synthesis of Mg-protoporphyrin-IX from exogenous protoporphyrin-IX, in a crude plastid pellet extracted from greening cucumber cotyledons was found to require l-glutamate as a cofactor. It has now been shown that glutamate acts in the presence of contaminating mitochondria to provide an ATP regenerating system. With purified plastids, Mg chelatase is not stimulated by glutamate; instead, it requires a high concentration of ATP and is greatly stimulated by added phosphoenolpyruvate and pyruvate kinase. GTP, UTP, CTP, and ITP will not substitute for ATP. ADP in the absence of an ATP generating system is completely ineffective, whereas it is slightly inhibitory in the presence of 10 mm ATP. AMP is strongly inhibitory in the reaction; 50% inhibition is obtained at approximately 3.5 mm AMP in the presence of 10 mm ATP.     

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities)

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.     

Protein Synthesis in Isolated Plastids during Chloroplast Development in Wheat

Light-driven protein synthesis in isolated plastids was studiedduring the greening of etiolated wheat (Triticum aestivum L.)seedlings. The process was divided into five phases (I to V)according to the recovery of plastids from the leaf tissue.The activity was not detected in the etioplasts, but rapidlyincreased to the maximum level in phase I and remained at thislevel through phase II. During the transition from phase IIto III, the activity rapidly decreased to one-third and thencontinued to decrease slowly. The plastid polypeptides synthesizedduring the greening were analyzed by SDS-polyacrylamide gelelectrophoresis. In phase I, membrane polypeptides having molecularweights of about 21k were synthesized, while 23 k membrane polypeptidewas synthesized in phases III, IV and V. Synthesis of solublepolypeptides of 50–60 k and membrane polypeptides of 15k and 30–35 k was active in phases I and II, but decreasedbetween phases II and III. (Received October 31, 1983; Accepted May 14, 1984)     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710