Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of ¹⁴C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO₂ fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO₂ assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.Abbreviations FBP Fructose-1,6-bisphosphate - HMP Hexose Monophosphates - PGA 3-phosphoglycerate - PMP Pentose Monophosphates - RBP Ribulose-1,5-bisphosphate - SBP Sedoheptulose-1,7-bisphosphate - TP Triose Phosphates     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation     

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     

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     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

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)     

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)     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

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.     

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