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     

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     

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     

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 mol·mg^–1·min^–1, 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.Abbreviation RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Effects of leaf age, nitrogen nutrition and photon flux density on the organization of the photosynthetic apparatus in leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves

Effects of leaf age, nitrogen nutrition and photon flux density (PFD) on the organization of the photosynthetic apparatus in leaves were investigated in a vine, Ipomoea tricolor Cav., which was grown horizontally so as to avoid mutual shading of leaves. The plants were grown hydroponically at two nitrate levels under two growth light treatments. For one group of the plants, leaves were exposed to full sunlight. For another group, respective leaves were artificially shaded in a manner that simulated changes in the light gradient with the development of an erect herbaceous canopy: old leaves were placed under progressively shadier conditions with growth of the plants (canopy-type shading). In all the treatments, chlorophyll (Chl) content gradually decreased with leaf age. Photosystem I (PSI) per Chl was constant, independent of leaf age, nitrogen nutrition and/or PFD. Photosystem II (PSII) and cytochrome / per Chl, and Chl a/b ratio were independent of leaf age and/or nitrogen nutrition but decreased with the decrease in growth PFD. Ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39, RuBPCase) per Chl steeply decreased with decrease in PFD. When leaves grown at the same PFD were compared, RuBPCase/Chl was lower in the plants grown under lower nitrogen availability and also decreased with leaf age in the plants grown without shading. These decreases were attributed to the curvilinear relationship between RuBPCase and Chl in leaves grown at full sunlight, that was independent of nitrogen availability and leaf age. From these results, it is concluded that the composition of the photosynthetic apparatus is independent of leaf age but changes depending on the light environment and total amount of photosynthetic components of the leaf.Abbreviations Chl chlorophyll - cyt f cytochrome f - PFD photon flux density - RuBPCase ribulose-1,5-bisphosphate carboxylase The author thanks Drs. K. Sonoike, Y. Kashino, K. Okada, H. Hatanaka, Y. Suzuki and A. Aoyama for technical advise. The author also thanks Drs. I. Terashima, A. Makino (Tohoku University, Sendai, Japan), Dr. J.R. Evans (Research School of Biological Sciences, Australian National University, Canberra) and Prof. A. Watanabe for valuable suggestions.     

Heat modification of ribulose-1,5-bisphosphate carboxylase/oxygenase by temperature pretreatment of wheat (Triticum aestivum L.) seedlings

The effect of low-, ambient- and high-temperature pretreatments (48 h at 4° C, 20° C or 36° C) of wheat seedlings (spring wheat Triticum aestivum L., cv. Kolibri) on the solubility properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase; EC 4.1.1.39) was studied. The extractable protein moiety of heat-pretreated plants exhibited increased solubility in dilute buffer (50 mM k-phosphate, pH 6.8), compared with protein extracted from 4° C- or 20° C-plants. The salting-out characteristics for ammonium-sulfate precipitation confirmed this finding since a delayed precipitation of extractable protein from 36°C-plants was observed. Using polyacrylamide gel electrophoresis, the in-vivo temperature-induced differences in protein solubility could be traced back to a change in the solubility of RuBPCase. The RuBPCase was purified from wheat seedlings, and the purified enzyme also exhibited differential solubility. In order to evaluate this further, purified RuBPCase was subjected to probing for conformational properties. A decrease of fluorescence of the RuBPCase 1-anilino-8-naphtalene sulfonate complex revealed that the RuBPCase from 36° C-plants had a more hydrophilic protein surface. Titration of the sulfhydryl groups of native RuBP-Case with 5,5-dithiobis (2-nitrobenzoic acid) pointed to a reduced accessibility of the R-SH groups in the case of the 36° C-type of RuBPCase. The large subunit of RuBPCase from 4° C/20° C-plants tended to give rise to an artificial lower-molecular-weight polypeptide which could not be found in crude or purified RuBPCase from heat-pretreated wheat seedlings.Abbreviations ANS 1-anilino-8-naphtalene sulfonate - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bis-phosphate carboxylase/oxygenase - RuBP ribulose-1,5-bis-phosphate     

Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange

The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective K _m and V _max values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the V _max values between the two species were greater. When the net activity of CO₂ exchange was calculated at the physiological CO₂-O₂ concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO₂-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO₂-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO₂ pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO₂ assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic - PAR photosynthetically active radiation - RuBP ribulose-1,5-bisphosphate     

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     

The relationship between carbon-dioxide-limited photosynthetic rate and ribulose-1,5-bisphosphate-carboxylase content in two nuclear-cytoplasm substitution lines of wheat,and the coordination of ribulose-bisphosphate-carboxylation and electron-transport capacities

Photosynthesis in two cultivars of Triticum aestivum was compared with photosynthesis in two lines having the same nuclear genomes but with cytoplasms derived from T. boeoticum. The in-vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) isolated from lines with T. boeoticum cytoplasm was only 71% of that of normal T. aestivum. By contrast, the RuBPCase activities calculated from the CO₂-assimilation rate at low partial pressures of CO₂, p(CO₂), were the same for all lines for a given RuBPCase content. This indicates that both types of RuBPCase have the same turnover numbers in-vivo of 27.5 mol CO₂·(mol enzyme)^–1·s^–1 (23°). The rate of CO₂ assimilation measured at normal p(CO₂), p _a =340 bar, and high irradiance could be quantitatively predicted from the amount of RuBPCase protein. The maximum rate of RuBP regeneration could also predict the rate of CO₂ assimilation at normal ambient conditions. Therefore, the maximum capacities for RuBP carboxylation and RuBP regeneration appear to be well-balanced for normal ambient conditions. As photosynthetic capacity declined with increasing leaf age, the capacities for RuBP carboxylation and RuBP regeneration declined in parallel.Abbreviations PAR photosynthetically active radiation - RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The flag leaf of wheat was examined for changes in quantity and activity of ribulose-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39), in the proteolytic degradation of RuBPCase and other native proteins, and in the ultrastructure of the leaf cells during grain development. Proteolytic degradation of RuBPCase at pH 4.8 increased until 8–10 d after anthesis, then declined, and increased again 16–18 d after anthesis. The second peak coincided with the onset of a preferential loss of immunologically recognizable RuBPCase. The specific activity and number of active sites per molecule of RuBPCase did not change during senescence. Examination of ultrastructure with the electron microscope showed little change in the appearance of the mitochondria as the flag leaf aged. Prominent cristae were still evident 35 d after anthesis. In contrast, the chloroplasts showed a progressive disruption of the thylakoid structure and an increasing number of osmiophilic glubules. The double membrane envelope surrounding the chloroplast appeared intact until at least 20 d after anthesis. The tonoplast also appeared intact up to 20 d. At later stages of senescence of the leaf the outer membrane of the chloroplast adjacent to the tonoplast appeared to break but the inner membrane of the envelope appeared intact until at least 35 d after anthesis.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase (EC. 4.1.1.39) I=Waters et al. 1980     

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     

Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L.

The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (Pisum sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N⁶-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-1,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis     

Variation in photosynthetic electron transport capacity in vivo and its effects on the light modulation of ribulose bisphosphate carboxylase

Photosynthetic electron transport capacity was varied in vivo in sugar beets using iron deficiency, and its effects on the light modulation of ribulose bisphosphate carboxylase (RuBPCase) studied. Three treatment groups corresponding to decreasing amounts of thylakoids per leaf area were examined: iron sufficient (control), moderately iron-stressed, and severely iron-stressed. Reduction in electron transport capacity in vivo was correlated with a substantial decrease in the level of RuBPCase activation, even at saturating irradiances. These results indicate a direct relationship between RuBPCase activation and photosynthetic electron transport. In addition, our data suggest that the activation of RuBPCase could not solely account for the increases in the photosynthetic rate at high irradiances; RuBPCase reached maximal activation at irradiances well below light saturation for net photosynthesis.Abbreviations Chl chlorophyll - FeCN ferricyanide - FBPase fructose 1,6-bisphosphatase - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose 1,5-bisphosphate carboxylase - SBPase sedoheptulose 1,7-bisphosphatase     

Variation in Ribulose 1,5 Bisphosphate Carboxylase Content in a Range of Winter Wheat Genotypes

Pyke, K A. and Leech, R. M. 1985. Variation in nbulose 1, 5bisphosphate carboxylase content in a range of winter wheatgenotypes. J. exp. Bot. 36: 1523–1529. Amounts of ribulose 1, 5 bisphosphate carboxylase (RuBPCase;E.C 4.1.1.39 [EC] ) were measured in the first leaves of 14 hexaploidwheat genotypes. The genotypes were representative of winterwheat grown in Britain during the past 150 years. The highest levels of RuBPCase per unit leaf area were foundin semi-dwarf genotypes which had more mesophyll cells per unitleaf area and smaller cells than tall genotypes. There was nosignificant correlation relating the year of introduction ofgenotypes to either the amount of RuBPCase per leaf or the amountper mesophyll cell Semi-dwarf genotypes tended to have smallerleaves and were less variable. Genotypic variation in the cellular content of RuBPCase is discussedin terms of genotypic differences in leaf development and thepotential for maximal RuBPCase accumulation. Key words: —Ribulose bisphosphate carboxylase, semi-dwarf wheat, cell size     

Conversion of ribulose-1,5-bisphosphate carboxylase to an acidic and catalytically inactive form by extracts of osmotically stressed Lemna minor fronds

The fronds of Lemna minor L. respond to a number of stresses, and in particular to an osmotic stress, by producing an enzyme system which catalyzes the oxidation of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) to an acidic and catalytically inactive form. During the first 24 h of osmotic stress the induced oxidase system does not seem to exert a significant in-vivo effect on RuBPCase, presumably because of compartmentation. Subsequently, the oxidase system gains access to the enzyme and converts it to the acid and catalytically inactive form and eventually the oxidase system declines in activity.A number of partially acidified forms of RuBPCase are formed during oxidation, and this process appears to be correlated with the disappearance of varying numbers of SH residues. The number of-SH residues in RuBPCase from Lemna has been estimated at 89. However, RuBPCase isolated from 24-h osmotically stressed fronds showed a reduction in the number of-SH residues per molecule from 89 to 54. It seems likely that the oxidation of-SH groups is causally related to the acidification of RuBPCase which occurs during osmotic stress.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - FPLC fast protein liquid chromatography - PMSF phenylmethylsulphonyl fluoride - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulfate     

Immunocytological and chemical studies on the stromacentre-forming protein from Avena plastids

The stromacentre (SC), a particular structure in the plastids of Avena, was isolated from etioplasts of Avena sativa by density gradient centrifugation and then analyzed and compared with ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) from A. sativa, with pyrenoids of Chlorella vulgaris and with the stromacentre of Opuntia. Purified SC-elements consisted of protein subunits with a relative molecular weight of 63 kDa, different from the isolated RuBPCase of A. sativa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After peptide mapping, the proteolytic cleavage patterns of the 63-kDa protein were also found to be different from those of the RuBPCase. Antibodies against SC-elements, RuBPCase, and the large subunit of RuBPCase were produced. Ouchterlony double immunodiffusion tests did not give crossreactions between the SC-elements and RuBPCase or the large subunit of this enzyme. Immunogold labelling of ultrathin sections showed that antibodies against the SC-elements marked the stromacentre in Avena, but not the pyrenoids in Chlorella. Antibodies against the large subunit of RuBPCase, however, did not label the SC, but labelled the stroma of the plastids in Avena and the pyrenoids of Chlorella. In Opuntia, a comparable structure described as an SC was not labelled by any of the antisera. Immunoelectrophoretical investigations demonstrated a strong correlation between the presence of the 63-kDa protein and the occurrence of the SC in different Avena species with and without SC.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SC stromacentre - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethy;)-1,3-propanediol Dedicated to Professor Ludwig Bergmann on the occasion of his 60th birthday     

Differential expression of the genes for ribulose-1,5-bisphosphate carboxylase and light-harvesting chlorophyll a/b protein in the developing barley leaf

The expression of genes in particular for light-harvesting chlorophyll a/b protein (LHCP) and ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been studied in the developing barley leaf. This has been done by analysis of the occurrence of both proteins within the different regions (1 to 6, beginning from the base) of the primary 7-day-old leaf. It has been found that LHCP already appears in the base of the leaf, whereas RuBPCase is primarily expressed in the apical expanding part of the leaf. The distribution of the mRNAs for both proteins within this gradient is in accordance with that of the proteins themselves, indicating that gene expression is not regulated at the level of translation in both cases. The poly(A) mRNA for LHCP occurs mainly in the basic sections 2 and 3, whereas that for RuBPCase is found throughout the leaf but primarily in the apical sections of the leaf.Abbreviations LHCP light-harvesting chlorophyll a/b protein - RuBPCase ribulose-1,5-bisphosphate carboxylase - TCA trichloroacetic acid     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl