The effects of light quality and intensity on the synthesis of ribulose-1,5-bisphosphate carboxylase and its mRNAs in the green alga Chlorogonium elongatum

In the green alga Chlorogonium elongatum the promoting effect of light on the synthesis of ribulose bisphosphate carboxylase/oxygenase (RuBPCase) is mainly caused by blue light of wavelengths between 430 nm and 510 nm, with a maximum effect at about 460 nm. Blue light also causes an increase in the amounts of the mRNAs for the large and the small subunits of the enzyme. Furthermore, the concentration of RuBPCase is affected by the light energy fluence rate. The rate of synthesis as well as the maximal obtainable concentration of the enzyme are functions of the light energy fluence rate up to 26 W·m^-2. No further increase occurs beyond that intensity. The quantity of irradiation also alters the concentrations of the subunit mRNAs. The results indicate that the changes in the mRNA levels are the major regulatory steps in the light-dependent synthesis of the RuBPCase enzyme.Abbreviations LSU large subunit - pSSU precursor of the small subunit - RuBPCase ribulose bisphosphate carboxylase/oxygenase EC 4.1.1.39 Dedicated to Prof. Dr. E. Bünning on the occasion of his 80 th birthday     

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     

Photo- and Metabolite Regulation of the Synthesis of Ribulose Bisphosphate Carboxylase/Oxygenase and the Phycobiliproteins in the Alga Cyanidium caldarium

In the eukaryotic and unicellular alga Cyanidium caldarium the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and the light gathering proteins phycocyanin (PC) and allophycocyanin (APC) is under the control of light and glucose, which is a metabolizable carbon source for this organism. Light promotes the synthesis of these proteins while glucose has a strong inhibitory effect on this process. All subunits of the proteins mentioned above are in vitro translation products of poly (A)⁻-RNA (Steinmüller, Kaling, Zetsche 1983 Planta 159: 308-313). Both factors—light and glucose—exert their effects mainly by modulation of the level of translatable messenger RNA for these proteins. Under autotrophic growth conditions the level of translatable RuBPCase-, PC-, and APC-messenger RNA is high, whereas in the presence of glucose the level of these mRNAs is low or not detectable at all.     

Regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase and its subunits in the flagellate Chlorogonium elongatum. Different levels of translatable messenger RNAs for the large and the small subunits in autotrophic and heterotrophic cells as determined by immunological techniques

Poly(A)-rich and poly(A)-free RNAs were isolated from autotrophic and heterotrophic cells of the phytoflagellate Chlorogonium elongatum and translated in an mRNA-depleted reticulocyte lysate system. Immunoprecipitation methods were improved to detect large and small subunits of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase synthesized in vitro. Large-subunit polypeptides were shown to be the translation products of poly(A)-free RNA having the same molecular weight as large subunits made in vivo. Small-subunit polypeptides were synthesized when poly(A)-rich RNA was used as a template. They were made in vitro as a precursor, with an Mr about 6000 larger than mature small subunits. Cells growing heterotrophically in the dark with acetate are provided with lower levels of mRNA activities for the large and the small subunits is at least partially controlled by the amounts of translatable mRNAs.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase expression in melon plants infected with Colletotrichum lagenarium

Ribulose-1,5-bisphosphate carboxylase/oxygelase (RuBPCase) was studied in melon leaves infected by Colletotrichum lagenarium, a fungal pathogen of melons. Electrophoretic analysis of melon leaf proteins indicated a strong effect of infection on RuBPCase, the subunits of which gradually disappeared during the different stages of infection. Enzyme activity also declined 4 d after inoculation and its content, measured by immunoelectrophoresis, decreased to a similar extent. Synthesis of the large and small subunits of RuBPCase was followed by in-vivo pulse-labeling experiments. A drastic decrease in the rate of RuBPCase-subunit synthesis occurred 3 d after inoculation and preceded the appearance of disease symptoms. There was an apparent coordination of the synthesis of the two subunits under these conditions.Abbreviations LS (SS) Large (small) subunit of RuBPCase - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Ultrastructural detection of ribulose-1,5-bisphosphate carboxylase protein and its subunit mRNAs in wild-type and holoenzyme-deficient Nicotiana using immuno-gold and in-situ-hybridization techniques

In-situ-localization techniques have been adapted to the ultrastructural detection of the holoenzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) and its composite large- and smallsubunit mRNAs in wild-type and mutant RuBPCase deficient plantlets of Nicotiana tabacum L. Immuno-gold techniques which show the distribution of target proteins have confirmed visually the presence of the holoenzyme in the wild-type plastids and its total absence in the enzyme-less mutant. Using in-situ hybridization coupled with electron microscopy and biotinylated probes for the two subunits, we have directly visualized specific small-subunit mRNAs located in the cytoplasm and large-subunit mRNAs confined to plastids in the enzyme-deficient mutant, and with apparent distributions comparable to those visualized in the wild-type counterpart. These results show that (i) gene products can be visualized in situ by electronmicroscopy techniques under conditions where the respective cellular compartments are readily recognizable and (ii) that an accumulation of mRNAs corresponding to the composite subunits can occur without translation and-or assembly of the protein.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU RuBPCase small subunit - LSU RubBPCase large subunit     

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae : Physical and Immunological Characterizations

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N⁶-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-M_r component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CF1 coupling-factor 1 of chloroplast ATPase - LHCP light-harvesting chlorophyll a/b-binding protein - LSU large subunit of Rubisco - NADPH-protochlorophyllide oxidoreductase Pchlide reductase - SDS sodium dodecyl sulfate - SSU small subunit of Rubisco We thank K. Hutchison (Dept. of Biochemistry, University of Maine, Orono, Maine, USA) and P. Gustafsson (Dept. of Plant Physiology, University of Umeå, Sweden) for providing the Larix and Pinus clones, and M. Ryberg (Dept. of Plant Physiology, University of Göteborg, Sweden), R. Ölmüller (Botanisches Institut, Universität München, FRG) and W. Lockau (Institut für Botanik, Universität Regensburg, FRG), for the gift of antisera towards Pchlide reductase, RuBPCase and LHCP, and ATPase, respectively. Supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council.     

Synthesis and assembly of bacterial and higher plant Rubisco subunits in Escherichia coli

The synthesis in Escherichia coli of both the large and small subunits of cereal ribulose bisphosphate carboxylase/oxygenase has been obtained using expression plasmids and bacteriophages. The level and order of synthesis of the large and small subunits were regulated using different promoters, resulting in different subunit pool sizes and ratios that could be controlled in attempts to optimize the conditions for assembly. Neither assembly nor enzyme activity were observed for the higher plant enzyme. In contrast, cyanobacterial large and small subunits can assemble to give an active holoenzyme in Escherichia coli. By the use of deletion plasmids, followed by infection with appropriate phages, it can be demonstrated that the small subunit is essential for catalysis. However, the small subunit is not required for the assembly of a large subunit octomer core in the case of the Synechococcus enzyme; self-assembly of the octomer will occur in an rbcS deletion strain. The cyanobacterial small subunits can be replaced by wheat small subunits to give an active enzyme in Escherichia coli. The hybrid cyanobacterial large/wheat small subunit enzyme has only about 10% of the level of activity of the wild-type enzyme, reflecting the incomplete saturation of the small subunit binding sites on the large subunit octomer, and possibly a mismatch in the subunit interactions of those small subunits that do bind, giving rise to a lower rate of turnover at the active sites.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - L large subunit - Rubisco ribulose bisphosphate carboxylase/oxygenase - S small subunit     

Regulatory factors involved in gene expression (subunits of ribulose-1,5-bisphosphate carboxylase) in mustard (Sinapis alba L.) cotyledons

Phytochrome-controlled appearance of ribulose-1,5-bisphosphate carboxylase (RuBP-Case) and its subunits (large subunit LSU, small subunit SSU) was studied in the cotyledons of the mustard (Sinapis alba L.) seedling. The main results were as follows: (i) Control of RuBPCase appearance by phytochrome is a modulation of a process which is turned on by an endogenous factor between 30 and 33 h after sowing (25° C). Only 12 h later the process begins to respond to phytochrome. (ii) The rise in the level of RuBP-Case is the consequence of a strictly coordinated synthesis de novo of the subunits. (iii) While the levels of translatable mRNA for SSU are compatible with the rate of SSU synthesis the relatively high LSU mRNA levels are not reflected in the rates of in-vivo LSU or RuBPCase syntheses. (iv) Gene expression is also abolished in the case of nuclear-encoded SSU if intraplastidic translation and concomitant plastidogenesis is inhibited by chloramphenicol, pointing to a plastidic factor as an indispensable prerequisite for expression of the SSU gene(s). (v) Regarding the control mechanism for SSU gene expression, three factors seem to be involved: an endogenous factor which turns on gene expression, phytochrome which modulates gene expression, and the plastidic factor which is an indispensable prerequisite for the appearance of translatable SSU mRNA.Abbreviations CAP chloramphenicol - cFR continuous farred light - LSU large subunit of RuBPCase - NADP-GPD NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) - Pfr far-red-absorbing form of phytochrome - pSSU precursor of SSU - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - SSU small subunit of RuBPCase     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of acetate and formate in continuous culture

Growth of Pseudomonas oxalaticus in carbon- and energy-limited continuous cultures with mixtures of acetate and formate resulted in the simultaneous utilization of both substrates at all dilution rates tested. During growth on these mixtures, acetate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and on the ratio of acetate and formate in the medium reservoir. At fixed acetate and formate concentrations in the inflowing medium of 30 and 100 mM, respectively, and dilution rates above 0.10h^-1, the severe repression of autotrophic enzymes resulted in a marked increase in bacterial dry weight compared to the growth yield of the organisms on the two substrates separately. Also, at these dilution rates a significant increase in isocitrate lyase activity was observed in the cells as compared to growth on acetate alone. This indicated that under these conditions more acetate was assimilated and less dissimilated since acetate was partly replaced by formate as the energy source. When formate was added to the reservoir of an acetate-limited culture (S_R=30 mM), derepression of RuBPCase synthesis was observed at formate concentrations of 50 mM and above. Below this concentration formate only served as an energy source for acetate assimilation; when its concentration was increased above 50 mM a progressively increasing contribution of carbon dioxide fixation to the total carbon assimilation was observed as the activity of RuBPCase in the cells increased. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic carbon dioxide fixation via the Calvin cycle is regulated by a repression/derepression mechanism.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

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.     

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO₃⁻ per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O₂ per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.     

Rapid variations in the content of the RNA of the small subunit of ribulose-1,5-bisphosphate carboxylase of mature tobacco leaves in response to localized changes in light quantity. Relationships between the activity and quantity of the enzyme

Mature green leaves from tobacco (Nicotiana tabacum L.) plants were submitted to contrasting light conditions; half of each leaf was shaded (changed from 60 to 25 mol photons· m^-2 ·s^-1=LL) and the other half was exposed to higher light (changed from 60 to 360 mol·m^-2· s^-1=HL) for 24 h. The activity and quantity of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were measured during the first 24 h in each leaf region and the variation was compared with that of small subunit (SSU)-and large subunit (LSU)-mRNA contents determined by a hybridot technique. Each leaf half responded separately to the actual light received. The activity of RuBPCase increased progressively in the HL zones and decreased in the LL zones. The RuBPCase-protein content was not significantly modified during the first 24 h but SSU-mRNA content responded very rapidly to the treatment. Within 2 h a significant difference in SSU mRNA appeared between LL and HL zones: at the end of the photoperiod the content in LL zones was approx. 25% of the initial value. The increase in the exposed zone, however, was not significant, indicating that there was a dissymmetry of the response to variation in incident white light. The LSU-mRNA contents from the same leaf extracts were totally unaffected by the light treatment. No day-night variations were noted in either SSU or LSU mRNAs in control plants.Abbreviation HL high-light irradiance - LL lower-ligh irradiance - LSU large subunit of RuBPCase - RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU small subunit of RuBPCase     

Amino-acid-transport mutant of Nicotiana tabacum L.

The structure of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) has been investigated by tilted-view electron microscopy of negatively stained monolayer crystals and image processing. The structure determined consists of a cylinder of octagonal cross-section with a large central hole. Based on this and other available evidence a model for the arrangement of the large and small subunits is suggested with the eight small subunits arranged equatorially around the core of eight large subunits.Abbreviations LS large subunit - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SS small subunit