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)     

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     

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     

Isolation and partial characterization of pyrenoids from the brown alga Pilayella littoralis (L.) Kjellm.

In order to isolate high yields of pyrenoids from the brown alga Pilayella littoralis it is necessary to pretreat them with 0.1% HgCl₂ in sea water for 3 h. Without this pretreatment there is a substantial loss of pyrenoid ground substance and yields are low. Pyrenoid fractions of high purity have been obtained using silica sol gradients. A partial characterization has shown the pyrenoid to be proteinaceous and lacking chlorophyll. SDS polyacrylamide gel electrophoresis has shown that the majority of protein present is accounted for by two polypeptides which resemble the large and small subunits of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39).Abbreviations DTT dithiothreitol - HEPES N-2-hydroxyethylniperazine N¹-2-ethanesulfonic acid - PEG polyethylene glycol - PVPP polyvinylpolypyrrolidone - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate     

Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus,as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - PEP phosphoenolpyruvate - RIE rocket immunoelectrophoresis - CIE crossed immunoelectrophoresis     

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     

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)     

Enzyme activities in an artificial stroma medium

When spinach leaf tissue was subjected to evaporative dehydration, photosynthetic capacity at very high (5%) CO₂ concentration and saturating irradiance (300 W·m^-2), decreased in parallel to the relative water content (RWC). A 50% inhibition was observed at 60–40% RWC. In order to examine whether the inhibition was caused by increased solute concentrations in chloroplasts or cytoplasm, an artificial stroma medium (ASM) was set up containing all major osmotically relevant solutes measured in isolated intact spinach chloroplasts. Subsequently, the response of enzyme activities to normal and to increased concentrations of ASM was examined. Inhibition of enzymes by a concerted increase of all solutes was well correlated to the in-vivo response of photosynthesis to dehydration (60% inhibition at double-strength ASM). Inhibitory solutes were mainly divalent inorganic anions, such as sulfate and phosphate. Inhibition of ribulose-1,5-bisphosphate carboxylase by these ions as studied in more detail. Inhibition of the enzyme by sulfate and phosphate was competitive with respect to ribulose-1,5-bisphosphate, but not with respect to CO₂. The K_I for sulfate was 2.1 mmol·l^-1 and for phosphate 0.57 mmol·l^-1. Sugars and amino acids at the concentrations found in spinach chloroplasts did not prevent inhibition of enzymes by anions. The results indicate that increased anion concentrations in cells and organelles are responsible for primary, quickly reversible effects of moderate dehydration on plant tissues.Abbreviations ASM artificial stroma medium - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate-carboxylase/oxygenase - RWC relative water content     

Variation in cellular ribulose-1,5-bisphosphate-carboxylase content in leaves of Triticum genotypes at three levels of ploidy

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 1.1.39) (RuBPCase) was quantified using polyacrylamide-gel electrophoresis in whole 9-d-old first leaves of 14 genotypes of Triticum, and cellular RuBPCase levels calculated. Diploids, tetraploids and hexaploids were analysed and it was confirmed that the RuBPCase level per cell is closely related to ploidy in wheat. Inter-genotypic variation in RuBPCase levels per cell and per leaf were surveyed. It was found that the interactions between leaf size, cell size and RuBPCase levels result in small variations in RuBPCase levels per unit leaf area between genotypes.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase     

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     

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     

Long-term chilling of young tomato plants under low light

The properties of two Calvin-cycle key enzymes, i.e. stromal fructose-1,6-bisphosphatase (sFBPase) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were studied in the cultivated tomato (Lycopersicon esculentum Mill.) and in four lines of a wild tomato (L. peruvianum Mill.) from different altitudes. During chilling for 14 d at 10°C and low light, the activation energy (E_A) of the reaction catalyzed by sFBPase decreased by 5–10 kJ·mol^–1 inL. esculentum and the threeL. peruvianum lines from high altitudes. InL. peruvianum, no loss or only small losses of enzyme activity were observed during the chilling. Together with the change in E_A, this indicates that the latter species is able to acclimate its Calvin-cycle enzymes to low temperatures. InL. esculentum, the chilling stress resulted in the irreversible loss of 57% of the initial sFBPase activity. Under moderately photoinhibiting chilling conditions for 3 d, theL. peruvianum line from an intermediate altitude showed the largest decreases in both the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and the in-vivo activation state of sFBPase, while the otherL. peruvianum lines showed no inhibition of sFBPase activation. Ribulose-1,5-bisphosphate carboxylase/oxygenase was isolated by differential ammonium-sulfate precipitation and gel filtration and characterized by two-dimensional electrophoresis. The enzyme fromL. esculentum had three isoforms of the small subunit of Rubisco, each with different isoelectric points. Of these, theL. peruvianum enzyme contained only the two more-acidic isoforms. Arrhenius plots of the specific activity of purified Rubisco showed breakpoints at approx. 17°C. Upon chilling, the specific activity of the enzyme fromL. esculentum decreased by 51%, while E_A below the breakpoint temperature increased from 129 to 189 kJ·mol^–1. In contrast, Rubisco from theL. peruvianum lines from high altitudes was unaffected by chilling. We tested several possibile explanations for Rubisco inactivation, using two-dimensional electrophoresis, analytical ultracentrifugation, gel filtration and inhibitor tests. No indications were found for differential expression of the subunit isoforms, proteolysis, aggregation, subunit disassembly, or inhibitor accumulation in the enzyme from chilledL. esculentum. We suggest that the activity loss in theL. esculentum enzyme upon chilling is the result of a modification of sulfhydryl groups or other sidechains of the protein.Abbreviations a.s.l. above sea level - Chl chlorophyll - DTT dithiothreitol - E_A activation energy - FBP fructose-1,6-bisphosphate - F_v/F_m ratio of variable to maximum chlorophyll fluorescence - HL high light (500 mol photons·m^–2·s^–1) - LSU large subunit of Rubisco - ME 2-mercaptoethanol - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - sFBPase stromal fructose-1,6-bisphosphatase - SSU small subunit of Rubisco     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

Pyrenoid protein from the brown alga Pilayella littoralis

Characterization by peptide mapping and amino acid analysis of the two major pyrenoid polypeptides from the brown alga Pilayella littoralis shows that they are very similar to the subunits of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) from this alga. The observed similarities are discussed in relation to previous pyrenoid protein characterization from members of the Chlorophyceae.Abbreviations DTT dithiothreitol - EDTA Na₂ ethylenediamine tetraacetic acid (disodium salt) - PMFS phenylmethylsul-phonylfluoride - PVPP polyvinylpyrrolidone - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TRIS 2-amino-2-(hydroxymethyl) propane-1,3-diol - TPCK L-1-tosylamido-2-phenylethylchoromethyl ketone     

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     

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     

Light intensity adaptation of the phycobiliprotein content of the red alga Porphyridium

In the unicellular red algae Porphyridium cruentum and P. aerugineum the phycobiliprotein content of the plastids is regulated by the applied energy fluence rate. Cells cultured at low energy fluence rates (220 W cm^-2) posses up to three times more phycobiliproteins than cells grown at high energy fluence rates (3200 W cm^-2). These values were obtained by direct measurement of the apoprotein of the phycobiliproteins. Transfer of cells from low to high energy fluence rates and vice versa results in an adaptation of the phycobiliprotein content to the new light conditions. This process starts immediately after the transfer of the cells and requires several days. On the other hand, the amount of the enzyme ribulose-1,5-bisphosphate carboxylase, which is also a prominent protein of the plastids of red algae, does not change significantly in response to differing fluence rates.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase     

Plastid ultrastructure and photosynthesis in greening petaloid hypsophylls

The ultrastructural and biochemicalphysiological aspects of postfloral greening have been studied in hypsophylls of Heliconia aurantiaca Ghiesbr., Guzmania cf. x magnifica Richter and Spathiphyllum wallisii Regel. In all three species the greening of the hypsophylls is due to plastid transformation, chloroplast formation proceeding from the initially different types of plastids. The degradation process of the original plastid structures and the mode of thylakoid formation are distinct in each case. In none of the species do the transformed plastids look identical to the chloroplasts of the corresponding foliage leaves. On a chlorophyll basis, the rate of photosynthesis of the greened hypsophylls surpasses the rate of the leaves considerably in Spathiphyllum, but is much lower in Heliconia (no data for Guzmania). In all species, anatomy, plastid structure, pigments, 77° K-fluorescence emission, ribulose-1,5-bis-phosphate carboxylase activities and short-term photosynthesis ¹⁴CO₂-assimilation patterns prove the greened hypsophylls to be capable of providing additional carbon to the developing fruits, thus supplementing the import of organic matter from the foliage leaves.Abbreviations MDH malate dehydrogenase (EC 1.1.1.37) - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Cold-hardening results in increased activity of enzymes involved in carbon metabolism in leaves of winter rye (Secale cereale L.)

Light- and CO₂-saturated photosynthesis of nonhardened rye (Secale cereale L. cv. Musketeer) was reduced from 18.10 to 7.17 mol O₂·m^–2·s^–1 when leaves were transferred from 20 to 5°C for 30 min. Following cold-hardening at 5°C for ten weeks, photosynthesis recovered to 15.05 mol O₂·m^–2·s^–1,comparable to the nonhardened rate at 20°C. Recovery of photosynthesis was associated with increases in the total activity and activation of enzymes of the photosynthetic carbon-reduction cycle and of sucrose synthesis. The total hexose-phosphate pool increase by 30% and 120% for nonhardened and cold-hardened leaves respectively when measured at 5°C. The large increase in esterified phosphate in coldhardened leaves occurred without a limitation in inorganic phosphate supply. In contrast, the much smaller increase in esterified phosphate in nonhardened leaves was associated with an inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose-phosphate synthase activation. It is suggested that the large increases in hexose phosphates in cold-hardened leaves compensates for the higher substrate threshold concentrations needed for enzyme activation at low temperatures. High substrate concentrations could also compensate for the kinetic limitations imposed by product inhibition from the accumulation of sucrose at 5°C. Nonhardened leaves appear to be unable to compensate in this fashion due to an inadequate supply of inorganic phosphate.Abbreviations DHAP dihydroxyacetone phosphate - Fru6P fructose-6-phosphate - Fru 1,6BP fructose-1,6-bisphosphate - Fru1,6BPase fructose-1,6-bisphosphatase - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - CH cold-hardened rye grown at 5°C - NH nonhardened rye grown at 24°C - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - UDPGlc uridine 5-diphosphoglucose This work was supported by operating grants from the Swedish Natural Sciences Research Council to G.Ö. and P.G.     

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