Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Intracellular localization of prenyltransferases of isoflavonoid phytoalexin biosynthesis in bean and soybean

The intracellular localization of prenyltransferases involved in the biosynthesis of the phytoalexins glyceollin in soybean (Glycine max L.) and phaseollin in French bean (Phaseolus vulgaris L.) has been investigated. By sucrose- and Percoll-gradient centrifugation of microsomes of an elicitor-challenged soybean cell culture, the membranes containing prenyltransferase were separated from the endoplasmic reticulum and shown to be lighter in density. In a continuous Percoll gradient the peak of prenyltransferase activity coincided with the peak of galactolipid synthesis, as determined by incorporation of uridine 5′-diphospho-[¹⁴C]galactose (UDP-[¹⁴C]galactose). Intact chloroplasts isolated from cupricchloride-treated bean leaves contained both prenyltransferase and UDP-galactose transferase activity. Both activities increased during chloroplast isolation. Fractionation of swollen chloroplasts on a discontinuous sucrose gradient showed prenyltransferase and UDP-galactose transferase activity in the envelope membrane subfraction. It is concluded that in both plants prenyltransferase is located in the envelope membrane of plastids. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts

Intact etioplasts with an intactness of 85% and with a cytosolic and a mitochondrial contamination of less than 10% were isolated from 8-d-old dark-grown barley (Hordeum vulgare) leaves. These plastids contained starch equivalent to 21.5 μmol of glucose per mg protein. From various likely precursors applied to isolated etioplasts, only dihydroxyacetone phosphate (DHAP) had significant effects on metabolite levels and on the internal ATP/ADP ratio. The concentration dependence of DHAP uptake exhibited saturation characteristics with half saturation at 0.36 mm DHAP and a maximal velocity of 6.6 μmol mg⁻¹ of protein h⁻¹. The transport was significantly inhibited by inorganic phosphate, pyridoxal-5′-phosphate, and 4,4′-diisothiocyano-2,2′-stilbenedisulfonate. The rate of glucose-6-phosphate uptake was much lower and not saturable up to a concentration of 10 mm. Exogenously applied [¹⁴C]DHAP was incorporated into starch at a rate of 0.14 μmol of DHAP mg⁻¹ of protein h⁻¹. Enzyme activities required to convert DHAP into starch were found to be present in etioplasts. Furthermore, enzymes generating ATP from DHAP for ADPglucose synthesis were also detected. Finally, a scheme is presented suggesting DHAP uptake to serve both as carbon skeleton and as energy source for starch synthesis, mediated by a translocator with properties similar to those of the triose phosphate translocator from chloroplasts.     

Sweet pepper plastids: enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane

Chloroplasts or chromoplasts were purified from sweet-pepper (Capsicum annuum L. cv. Yolo Wonder) fruits and analysed with respect to their enzymic equipment, the transport properties across the envelope membrane, and for the presence of a functional oxidative pentose-phosphate pathway (OPPP). It was demonstrated that both types of plastid contain enzyme activities that allow glycolysis and OPPP. During the developmental conversion from chloroplasts to chromoplasts the activities of enzymes catalysing potentially rate-limiting reactions in glycolysis increased considerably. Most enzyme activities involved in the plastidic OPPP stayed constant or decreased during ripening, but transaldolase activity increased by more than 500%. To analyse whether pepper fruit chromoplasts are able to use exogenously supplied carbohydrates for the OPPP we measured the rate of ¹⁴CO₂ release after application of radioactively labelled precursors. Isolated pepper fruit chromoplasts used exogenously supplied [U¹⁴C]glucose- 6-phosphate (Glc6P) as a precursor for the OPPP. The metabolic flux through this pathway was stimulated by the presence of additional compounds which require reducing equivalents for further conversion, e.g. nitrite, or 2-oxoglutarate plus glutamine. The [¹⁴C]Glc6P-driven OPPP in isolated chromoplasts exhibited saturation with rising concentrations of Glc6P, reaching highest rates at an external concentration of about 2 mM. Exogenously given [U¹⁴C]glucose 1-phosphate (Glc1P)′ did not lead to a release of ¹⁴CO₂, indicating that this hexose phosphate is not taken up into the intact plastid. Using a proteoliposome system in which the envelope membrane proteins from sweet-pepper chromoplasts were functionally reconstituted we demonstrated that Glc6P is transported in counter-exchange with inorganic phosphate (P_i) or other phosphorylated intermediates. The Glc6P was taken up into proteoliposomes with an apparent K _m of 0.34 mM. Surprisingly, in contrast to tomato fruit plastids, isolated chromoplasts from sweet-pepper fruits do not possess a phosphate translocator allowing the uptake of Glc1P. Rising exogenous concentrations of dihydroxyacetone phosphate strongly inhibited the metabolic flux through the OPPP. This observation is discussed with respect to the presence of two phosphate translocator proteins in the envelope of sweet-pepper chromoplasts and with respect to possible metabolic changes occurring in heterotrophic tissues during development. Received: 24 April 1997 / Accepted: 16 June 1997     

Comparison of Properties of the Proteolytic Degradation of Unassembled Nuclear-encoded Subunits of Ribulose-1,5-bisphosphate Carboxylase and of the Coupling Factor of Photophosphorylation CF1*

The proteolytic degradation of unassembled small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase and of the δ-subunit of the coupling factor of photophosphorylation CF₁ were analyzed and compared in vitro in the presence of stroma or membrane preparations from ribosome-deficient plastids isolated from 32°C-grown rye leaves (Secale cereale L.). Extracts obtained from 70S ribosome-deficient rye leaves after radioactive labeling were used as substrate source for the unassembled polypeptides. Soluble stroma as well as membrane preparations from isolated plastids contained proteolytic activities catalyzing the degradation of both the small subunits of ribulose-1,5-bisphosphate carboxylase and CF₁-δ in vitro. Maximal in vitro degradation was observed at pH 2–3 for the unassembled small subunits, but at pH 6–7 for the purified holoprotein of ribulose-1,5-bisphosphate carboxylase, and at pH 6.0 for unassembled CF₁-δ. Degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase at pH 3.0 was stimulated by Cu²⁺ but not by Ca²⁺, Mg²⁺ or ATP. At pH 3.0 the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase was not inhibited by various protease inhibitors but was even stimulated. At pH 7.0 its degradation was inhibited by HgCl₂ and diazoacetyl nor-leucine methyl ester + Cu-acetate. The degradation of CF₁-δ was markedly inhibited by phenylmethylsulphonyl fluoride (PMSF) and to a lesser extent by 1,10-phenanthroline. According to present results different proteolytic systems appear to be involved in the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase and of unassembled CF₁-δ.     

Uptake of isopentenyl diphosphate by plastids isolated from Vitis vinifera L. cell suspensions

The uptake of [1-¹⁴C]isopentenyl diphosphate by intact plastids purified from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan was investigated using vacuum-filtration and silicone-oil-filtering techniques. Transport across the plastid envelope which was stimulated by cations, such as Mg²⁺ and Mn²⁺, was characterized by a K _m of approx. 0.5 mM and a V _max of 25 nmol·(mg protein)^-1·-h^-1. The data showed that isopentenyl diphosphate apparently accumulated in the plastid against a concentration gradient. The involvement of a protein carrier was suggested by the strong inhibition of the uptake by compounds which are known to block SH groups. Thus, the saturation kinetics together with the pH optimum (7.5–8), the temperature dependence (maximum incorporation at 37 °C) and the competitive inhibition by a structural analogue of the substrate (aminophenylethyl diphosphate) provided evidence for a mechanism of uptake by facilitated diffusion. The carrier identified may thus play a major role in supplying the plastid compartment with isopentenyl diphosphate for isoprenoid biosynthesis.Abbreviations APP aminophenylethyl diphosphate - DMAPP dimethylallyl diphosphate - GPP geranyl diphosphate - IPP isopentenyl diphosphate - NEM N-ethylmaleïmide - PCMB p-chloromercuribenzoate     

Carotenoid and chlorophyll biosynthesis in isolated plastids from mustard seedling cotyledons (Sinapis alba L.) during etioplast-chloroplast conversion

Etioplasts and etiochloroplasts, isolated from seedlings of white mustard (Sinapis alba L.) grown in continuous far-red light, and chloroplasts isolated from cotyledons and primary leaves of white-light-grown seedlings exhibit high prenyl-lipid-forming activities. Only the etioplasts and etiochloroplasts, and to a much lesser extent chloroplasts from cotyledons are capable of forming carotenes from isopentenyl diphosphate as substrate, whereas in chloroplasts from primary leaves no such activities could be detected. By subfractionation experiments, it could be demonstrated that the phytoene-synthase complex in etioplasts and etiochloroplasts is present in a soluble form in the stroma, whereas the subsequent enzymes, i.e. the dehydrogenase, cis-trans isomerase and cyclase are bound to both membrane fractions, the prolamellar bodies/prothylakoids and the envelopes. In good agreement with previous results using isolated chromoplasts and chloroplasts, it is concluded that the phytoene-synthase complex may change its topology from a peripheral membrane protein in non-green plastids to a tightly membrane-associated protein in chloroplasts. This change is apparently paralleled by altered functional properties which render the complex undetectable in isolated chloroplasts. Further experiments concerning the reduction of chlorophyll a containing a geranylgeranyl side chain to chlorophyll a indicate that the light-induced etioplast-chloroplast conversion is accompanied by a certain reorganization of the polyprenoid-forming enzymatic equipment.Abbreviations Chl a chlorophyll a - Chl_GG chlorophyll a containing a geranylgeranyl side chain - HPLC high-performance liquid chromatography - Tris 2-ammo-2-(hydroxymethyl)-1,3-propanediol     

Investigation of the site of synthesis of chIcoroplastic enzymes of nitrogen metabolism by the use of heat-treated 70S ribosomedeficient rye leaves

The activities of the enzymes nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.7.1), glutamate-oxaloacetate aminotransferase (EC 2.6.1.1), and glutamate dehydrogenase (EC 1.4.1.2) were compared in light-grown green or etiolated leaves of rye seedlings ( Secale cereale L. cv. Halo) raised at 22°C, and in the bleached 70S ribosome-deficient leaves of rye seedlings grown at a non-permissive high temperature of 32°C. Under normal permissive growth conditions the activities of most of the enzymes were higher in light-grown, than in dark-grown, leaves. All enzyme activities assayed were also observed in the heat-treated 70S ribosome-deficient leaves. Glutamine synthetase, glutamate synthase, and glutamate-oxaloacetate aminotransferase occurred in purified ribosome-deficient plastids separated on sucrose gradients. For glutamate-oxaloacetate aminotransferase four multiple forms were separated by polyacrylamide gel electrophoresis from leaf extracts. The chloroplastic form of this enzyme was also present in 70S ribosome-deficient leaves. It is concluded that the chloroplast-localized enzymes nitrite reductase, glutamine synthetase, glutamate synthase and glutamate-oxaloacetate aminotransferase, or their chloroplast-specific isoenzyme forms, are synthesized on cytoplasmic 80S ribosomes.     

Prenyl lipid and fatty-acid synthesis in isolatedAcetabularia chloroplasts

A gentle procedure allowed the isolation of intact and highly active chloroplasts from the unicellular green algaAcetabularia mediterranea. These chloroplasts incorporated carbon from NaH¹⁴CO₃ into fatty acids and prenyl lipids at a rate of about 20–50 nmol carbon· (mg chlorophyll)⁻¹·h⁻¹. Most of the fatty acids formed in vitro were esterified in galactolipids. The main prenyl lipids synthesized were the chlorophyll side chain, intermediates of the carotenogenic path, α-and β-carotene, as well as lutein. Large amounts of [1-¹⁴C]acetate were incorporated, but exclusively into fatty acids.Isopentenyl diphosphate was a good substrate for prenyl-lipid formation in hypotonically treated chloroplasts. The envelope of intact chloroplasts, however, was impermeable to this compound. Intermediates of the mevalonate pathway were not accepted as precursors under conditions whereisopentenyl diphosphate was well incorporated. The results show that the lipid biosynthetic pathways in the plastids ofAcetabularia, a member of the ancient family of Dasycladaceae, are very similar to those in higher-plant plastids. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

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.

     

Specific Transport of Inorganic Phosphate and C3- and C6-Sugar-Phosphates across the Envelope Membranes of Tomato (Lycopersicon esculentum) Leaf-Chloroplasts,Tomato Fruit-Chloroplasts and Fruit-Chromoplasts

Plastidial envelope membranes were isolated from tomato (Lycopersicon esculentum) leaves and green and red tomato fruits by isopycnic discontinuous sucrose density gradient centrifugation. Solubilized envelope membrane proteins were reconstituted into liposomes. Transport measurements revealed that the phosphate translocator from tomato leaves transports inorganic phosphate, 3-phosphoglycerate and triosephosphates. The phosphate translocators of green and red fruit plastids catalyze, in addition to the transport of these substrates, also the transport of glucose-6-phosphate, glucose-1-phosphate and phosphoenolpyruvate.     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids

Single senescent leaves attached to decapitated shoots of Nicotiana rustica L. regreened, especially when treated with cytokinin. Regreening caused an increase in leaf thickness, due to cell expansion. Senescent leaf plastids (gerontoplasts) were smaller than green chloroplasts, with degenerated membrane systems and stroma, and larger plastoglobuli. At advanced senescence, micrographs showed disintegrating gerontoplasts, reduced numbers of plastids were counted, and regreening became variable. The redevelopment of grana and stroma in regreening plastids was accelerated by cytokinin. All plastids in regreening leaves were identifiable as redifferentiating gerontoplasts because of their content of plastoglobuli and starch. Immunogold labelling showed significant association of POR with etioplasts in cotyledons, but with mature plastids in regreening leaves. No proplastids or dividing chloroplasts were observed in regreening leaves. Plastids numbers declined during senescence and did not increase again during regreening. It is concluded that the chloroplasts of regreening leaves arose by redifferentiation of gerontoplasts.Keywords: Chloroplasts, cytokinin, Nicotiana, senescence, regreening.      

Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.) cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex

Chloroplasts from the cotyledons of mustard (Sinapis alba L.) seedlings were isolated on Percoll gradients, and showed a high degree of intactness (92%) and purity as judged by electron microscopy and marker-enzyme analysis (cytoplasmic contamination lower than 0.4% on a protein basis). The chloroplasts synthesized longchain fatty acids from both precursors [1-¹⁴C] acetate and [2-¹⁴C]pyruvate; maximum incorporation rates were 96 nmol·(mg Chl)^-1·h^-1 for acetate and 213 nmol·(mg Chl)^-1·h^-1 for pyruvate. Acetyl-CoA-producing enzymatic activities, namely acetyl-CoA synthetase (EC 6.2.1.1.) and a pyruvate dehydrogenase complex, showed specific activities of 14.8 nmol·(mg protein)^-1·min^-1 and 18.2 nmol·(mg protein)^-1·min^-1, respectively. The glycolytic enzymes phosphoglyceromutase (EC 2.7.5.3) phosphopyruvate hydratase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were all found to be components of these chloroplasts, thus indicating a possible pathway for intraplastid acetyl-CoA formation.Abbreviations ACS acetyl coenzyme A synthetase - Chl chlorophyll - DTE 1,4-dithioerythritol - PDHC pyruvate dehydrogenase complex - 3-PGA 3-phosphoglyceric acid     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.