The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.     

CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

The mechanism of the control of carbon fixation by the pH in the chloroplast stroma

The salts of several weak acids have been used to render the envelope permeable to protons. In order to investigate the role of stromal pH changes in the light regulation of CO₂ fixation, formate, octanoate, nitrite, and glyoxylate have been tried as tools to reverse the light-dependent alkalization of the stroma. For this purpose, the decrease of the stromal pH in illuminated spinach chloroplasts, as caused by the addition of these substances or by instantaneous lowering of the pH in the medium, has been compared with the corresponding decrease of CO₂ fixation and the change of stromal metabolite levels. It appears from out data that formate and octanoate are suited best to obtain a specific inhibition of CO₂ fixation by lowering the stromal pH. The measurement of the corresponding metabolite levels indicates that this inhibition is primarily due to an inhibition of fructose- and sedoheptulose bisphosphatase. It is concluded that these two enzymes are important regulatory steps for the light control of CO₂ fixation.     

The effect of oxygen on 14CO2 fixation in mesophyll cells isolated from Digitaria sanguinalis (L.) Scop. leaves

Mesophyll cells were isolated from fully-expanded leaves of $\text{Digitaria sanguinalis}$ (L.) Scop. by a combined maceration-filtration technique. In the presence of pyruvate, photosynthetic ¹⁴CO₂ uptake in the isolated cells was not inhibited by atomospheric levels of oxygen. In contrast, superatmospheric levels of oxygen substantially inhibited the light-dependent fixation of ¹⁴CO₂. These oxygen effects are similar to those observed with intact C4 leaves and suggest that the lack of inhibition of C4 photosynthesis by atmospheric levels of oxygen results from the relative oxygen-insensitivity of the phosphopyruvate carboxylase-CO₂ pump in the mesophyll.     

The mechanism of the control of carbon fixation by the pH in the chloroplast stroma. Studies with nitrite-mediated proton transfer across the envelope.

1. CO2 fixation of intact spinach chloroplasts is inhibited by nitrite in a pH-dependent mode. At pH 7.3 in the medium 1 mM NaNO2 and at pH 7.9 5 mM NaNO2 were required for 50% inhibition. 2. The addition of nitrite leads to an acidificiation in the stroma. It appears that nitrite renders the envelope permeable for protons resulting in a breakdown of the pH gradient between the external space and the stroma. 3. In view of earlier results on the pH sensitivity of C02 fixation it is concluded that this pH shift in the stroma is responsible for the observed inhibition of CO2 fixation by nitrite. 4. Octanoate and to some extent also high concentrations of bicarbonate and acetate have a similar effect as nitrite in inhibiting CO2 fixation through an acidification in the stroma. 5. The levels of the intermediates of the CO2 fixation cycle were measured. A strong rise of the levels of fructose- and sedoheptulose biphosphates and a concomitant decrease of the corresponding monophosphates was observed during inhibition of CO2 fixation. It appears that the enzymatic steps of the CO2 fixation cycle responsible for the overall inhibition of CO2 fixation caused by lowering of the H+ concentration in the stroma are fructose- and sedopheptulose bisphosphatase. These two enzymes have an important function in the light regulation of CO2 fixation.     

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO₂ concentrations by employing a CO₂-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO₂-fixing enzyme, RubisCO. The efficiency of CO₂ fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO₂. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.     

Fermentative products and dark CO2 fixation during germination of seeds of Cicer arietinum

During the germination of Cicer arietinum L. the amounts of ethanol, lactate and malate reached their highest values at 24 hr, the concentration of ethanol being about 4 times that of lactate and twice that of malate. The activities of phosphoenolpyruvate carboxylase and malic enzyme seem to be correlated with the ability of cotyledons to fix CO₂ from NaH¹⁴CO₃ into malate and with the further decrease in this metabolise from 36 hr onwards.     

CO2和O3浓度倍增及其交互作用对大豆叶绿体超微结构的影响

应用透射电镜观察了模拟大气CO2和O3浓度倍增及其交互作用(开顶箱法)对大豆叶肉细胞叶绿体超微结构的影响。结果表明,CO2浓度倍增促进了大豆叶绿体的发育,内含淀粉粒积累明显增多、体积增大;叶绿体被膜保持完好;叶绿体基粒片层排列整齐,而O3浓度倍增抑制了叶绿体内淀粉粒的累积,并导致叶绿体被膜破碎,片层解体,严重地破坏了叶绿体的结构和功能CO2和O3浓度倍增的交互作用对叶绿体超微结构有不同程度的破坏,但二者浓度呈梯度增加对叶绿体的损害作用要大于二者浓度持续倍增对叶绿体的影响,进一步表明CO2正效应对O3负效应的补偿作用。     

Rapid decarboxylation of the products of dark fixation of CO2 in roots of pisum and Plantago

Seedlings of Pisum sativum and excised roots of Plantago major and P. lanceolata were given, in the dark, a pulse of ¹⁴CO₂ in air followed by a chase in ¹²CO₂ in air. A very substantial proportion of the ¹⁴C fixed into organic compounds in the pulse was lost from the tissues in the chase. The activity of NAD malic enzyme in extracts of roots of all three species exceeded their rate of respiration. Azide, 2-n-butylmalonate, and salicylhydroxamic acid each inhibited CO₂ fixation by excised roots of pea. The first two compounds inhibited respiratory gas exchange, but the third stimulated it. Arguments are presented for the widespread diversion of phosphoenolpyruvate from glycolysis to oxaloacetate and thence to malate in the cytosol followed by transport of the malate into the mitochondria for conversion to pyruvate via NAD malic enzyme. No differences, in the above respects, were found between the two species of Plantago.     

The critical role of the loops of triosephosphate isomerase for its oligomerization,dynamics, and functionality

Triosephosphate isomerase (TIM) catalyzes the reaction to convert dihydroxyacetone phosphate into glyceraldehyde 3‐phosphate, and vice versa. In most organisms, its functional oligomeric state is a homodimer; however, tetramer formation in hyperthermophiles is required for functional activity. The tetrameric TIM structure also provides added stability to the structure, enabling it to function at more extreme temperatures. We apply Principal Component Analysis to find that the TIM structure space is clearly divided into two groups—the open and the closed TIM structures. The distribution of the structures in the open set is much sparser than that in the closed set, showing a greater conformational diversity of the open structures. We also apply the Elastic Network Model to four different TIM structures—an engineered monomeric structure, a dimeric structure from a mesophile—Trypanosoma brucei, and two tetrameric structures from hyperthermophiles Thermotoga maritima and Pyrococcus woesei. We find that dimerization not only stabilizes the structures, it also enhances their functional dynamics. Moreover, tetramerization of the hyperthermophilic structures increases their functional loop dynamics, enabling them to function in the destabilizing environment of extreme temperatures. Computations also show that the functional loop motions, especially loops 6 and 7, are highly coordinated. In summary, our computations reveal the underlying mechanism of the allosteric regulation of the functional loops of the TIM structures, and show that tetramerization of the structure as found in the hyperthermophilic organisms is required to maintain the coordination of the functional loops at a level similar to that in the dimeric mesophilic structure.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts

Low concentrations of hydrogen peroxide strongly inhibit CO₂ fixation of isolated intact chloroplasts (50% inhibition at 10⁻⁵ M hydrogen peroxide). Addition of catalase to a suspension of intact chloroplasts stimulates CO₂ fixation 2–6 fold, indicating that this process is partially inhibited by endogenous hydrogen peroxide formed in a Mehler reaction.

The rate of CO₂ fixation is strongly increased by addition of Calvin cycle intermediates if the catalase activity of the preparation is low. However, at high catalase activity addition of Calvin cycle intermediates remains without effect. Obviously the hydrogen peroxide formed at low catalase activity leads to a loss of Calvin cycle substrates which reduces the rate of CO₂ fixation.

3-Phosphoglycerate-dependent O₂-evolution is not influenced by hydrogen peroxide at a concentration (5 · 10⁻⁴ M) which inhibits CO₂ fixation almost completely. Therefore the inhibition site of hydrogen peroxide cannot be at the step of 3-phosphoglycerate reduction. Dark CO₂ fixation of lysed chloroplasts in a hypotonic medium is not or only slightly inhibited by hydrogen peroxide (2.5 · 10⁻⁴ M), if ribulose-1,5-diphosphate, ribose 5-phosphate or xylulose 5-phosphate were added as substrates. However, there is a strong inhibition of CO₂ fixation by hydrogen peroxide, if fructose 6-phosphate together with triose phosphate are used as substrates. This indicates that hydrogen peroxide interrupts the Calvin cycle at the transketolase step, leading to a reduced supply of the CO₂-acceptor ribulose 1,5-diphosphate.     

高浓度二氧化碳对植物影响的研究进展

工业革命后全球大气CO2浓度持续上升,不仅对全球气候的变迁产生重大影响,而且对植物的形态、水分利用、蛋白质合成、光合、抗性、生长及生物量等都有不同程度的影响。高浓度CO2促进植物根、幼苗的生长,叶片增厚,降低气孔密度、气孔导度及蒸腾速率,增加水分利用效率、作物的产量及生物量,促进乙烯生物合成,增强植物的抗氧化能力。不同光合途径(C3、C4及CAM)及不同植被类型的植物对高浓度CO2的响应不同。长期和短期的高浓度CO2处理,植物响应方式有很大的差异,如短期高CO2处理使光合能力增强,而长期处理则使光合能力下调。     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO₂ fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO₂ fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO₂ fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO₂ also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO₂ fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.