Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q_B polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.     

Comparative analysis of the action of cytokinin and light on the formation of ribulosebisphosphate carboxylase and plastid biogenesis

The role of cytokinin in plastid biogenesis was investigated in etiolated rye leaves (Secale cereale L.) and compared with the effect of white light. Cytokinin deficiency of the leaves was induced by early excision of the seedling roots and reversed by the application of kinetin. The cytokinin supply had a much greater influence on plastid biogenesis than on leaf growth in general. The activities of several chloroplastic enzymes were increased 200%–400% after kinetin treatment of cytokinin-depleted leaves. The activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and the amount of fraction-I protein even showed a sevenfold increase. In cytokinin-depleted leaves the development of ribulose-1,5-bisphosphate carboxylase and NADP-glyceraldehydephosphate dehydrogenase was specifically, and markedly inhibited by actinomycin D. The inhibition was partially or even completely overcome after treatment with kinetin. However, under all conditions, RNA synthesis of the leaves, was only partially inhibited by actinomycin D. According to immunologic studies, all dark-grown leaves, in addition to the complete enzyme, contained an excess of free small subunit of ribulose-1,5-bisphosphate carboxylase that was absent in mature light-grown leaves. The most striking accumulation of free small subunit, protein occurred in cytokinin-depleted dark-grown leaves, indicating a deficiency of the plastidic synthesis of the large subunit. The capacity as well as the activity of plastidic protein synthesis was preferentially increased by cytokinin and light. Cytokinin increased, the amount of plastidic ribosomes per leaf and relative to the amount of cytoplasmic ribosomes. While the percentage of cytoplasmic ribosomes bound as polyribosomes was little affected by the cytokinin supply, the proportion of plastidic polyribosomes was increased from 11% to 18% after kinetin treatment of cytokinin-depleted leaves. In the light, the proportion of plastidic polyribosomes reached 39% of the total plastidic ribosomes.Abbreviations RuBP carboxylase ribulose-1,5-bisphosphate carboxylase - NADP-GAP dehydrogenase NADP-dependent glyceraldehyde-3-phosphate dehydrogenase     

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.     

Ribulose bisphosphate carboxylase synthesis in barley leaves: a developmental approach to the question of coordinated subunit synthesis

The coordination of the synthesis of the large and small subunits of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was studied in young light-grown barley (Hordeum vulgare L. var. UC566) leaves. Since a barley leaf is a continuum of different aged cells with the youngest cells at the base and the oldest at the tip, developmental changes could be investigated by comparing different leaf regions. The rate of total cytoplasmic protein synthesis increased to a maximum before the rate of total organelle protein synthesis. The different positions of the maxima suggested that the synthesis of the small RuBPCase subunit on cytoplasmic ribosomes and the large RuBPCase subunit on chloroplast ribosomes might not be coupled during barley leaf development. However, measurements of the amounts and rates of synthesis of the subunits showed that they were coupled. Although the amounts of the RuBPCase subunits increased from the younger to the older leaf regions, the subunits were present in an equimolar ratio. While the rates of synthesis of both subunits increased to a maximum in a midleaf region and then declined, the ratio of the rates remained constant. That the subunit amounts remained equimolar and the synthetic rates proportional while total RuBPCase synthesis was changing indicated that the synthesis of the subunits was closely coordinated during leaf development. A close coordination was also supported by the kinetics of the inhibition of subunit synthesis in the presence of cycloheximide.     

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     

Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase

Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO2 and Mg2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. Incubation of the carboxylase with potassium [14C] cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO2 binding sites on the enzyme.     

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.     

In vitro synthesis of the large subunit of ribulose diphosphate carboxylase on 70 S ribosomes

Polyribosomes isolated from greening barley leaves were active in directing protein synthesis, using soluble components isolated from Escherichia coli. A peptide of 55,000 molecular weight was a major product of translation activity. This peptide was precipitated by antibody to ribulose 1,5-diphosphate carboxylase (RuDPCase) and comigrated with the large subunit of RuDPCase on sodium dodecyl sulfate-polyacrylamide gels. Cyanogen bromide peptides of the peptide of 55,000 molecular weight also corresponded to the peptides prepared from authentic RuDPCase large subunit. The peptides synthesized were shown by sucrose density gradient sedimentation to be largely associated with 70 S ribosomes.     

In vivo chloroplast protein synthesis by the chromophytic alga Olisthodiscus luteus

Information on the ctDNA protein coding profile of the Chlorophyta, Rhodophyta, and Chromophyta might provide clues to the evolutionary mechanism(s) by which plants diverged into these three phylogenetic groups. The purpose of this study was to examine the ctDNA protein coding profile of the chromophytic plant Olisthodiscus luteus. Whole cells were labeled in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Control experiments demonstrate that the chloroplast proteins labeled in vivo by this technique form a distinct subset of the total proteins synthesized by the cell. Approximately 50 plastid proteins (35 soluble, 15 membrane) were detected after two-dimensional gel electrophoresis and fluorography. Three ctDNA-coded proteins, the large subunit of ribulosebisphosphate carboxylase, the apoprotein of the P700-chlorophyll a-protein complex, and the "photogene" were identified. These proteins are also coded by chlorophytic ctDNA. Unexpectedly, the ctDNA of Olisthodiscus was shown to code for the small subunit of ribulosebisphosphate carboxylase. The gene for this enzyme subunit is nuclear coded in all chlorophytic plants that have been analyzed.     

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.     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.     

Synthesis and degradation of unassembled polypeptides of the coupling factor of photophosphorylation CF1 in 70S ribosome-deficient rye leaves

The formation of polypeptides of the coupling factor CF1 was investigated in 70S ribosome-deficient rye leaves generated by growing the plants at a non-permissive elevated temperature of 32 degrees C, in order to analyse mechanisms coordinating subunit accumulation. Antibodies were raised in rabbits against total CF1 as well as against its five individual subunits purified from chloroplast thylakoids from rye leaves. Several immunological techniques applying these antibodies (immunoprecipitation, immunoblotting, antibody affinity chromatography) were unable to detect the presence of any of the CF1 subunits in heat-treated 70S ribosome-deficient leaves. After in vivo labeling with L-[35S]methionine and subsequent immunoprecipitation, however, radioactivity was found to be incorporated into the subunits gamma and delta, but not into alpha, beta and epsilon, in 70S ribosome-deficient leaves, demonstrating the cytoplasmic synthesis of CF1-gamma and CF1-delta. Chase experiments after in vivo labeling with L-[35S]methionine indicated that the unassembled subunits gamma and delta were rapidly and preferentially degraded, while they were stabilized when integrated into the complete CF1 complex in normal green leaves from permissive growth conditions. The apparent half-times of the unassembled subunits were 2 h for CF1-gamma and 4 h for CF1-delta in 32 degrees C-grown leaves. Several other, stromal, plastid proteins of cytoplasmic origin were stable in 32 degrees C-grown leaves during the period of chase. In etiolated leaves total CF1, including all subunits, appeared to be less stable than in green leaves grown under permissive temperature conditions in light. Rapid degradation of the excess of unassembled subunits is regarded as an important mechanism ensuring a constant stoichiometry and apparently synchronous development of CF1 subunits.     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.