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.     

Role of the phosphoinositide pathway in the light-dependent C4 phosphoenolpyruvate carboxylase phosphorylation cascade in Digitaria sanguinalis protoplasts

Stimulus-response coupling in animal cells frequently involves the hydrolysis of PtdIns(4,5)P(2) which is catalysed by phosphoinositide-specific phospholipase C (PI-PLC). There is an increasing body of evidence for PI-PLC-based signalling in plant cells; however, the physiological role of this system remains poorly documented in plants. Our data provide the first evidence that a PI-PLC-based signalling system is a committed step in the transduction chain controlling the phosphorylation state of C(4) phosphoenolpyruvate carboxylase (PEPC), the regulation of which is central to the assimilation of atmospheric CO(2) in C(4) plants.     

Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator

Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix ¹⁴C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations P_i orthophosphate - PLP pyridoxal 5-phosphate - TP triosephosphate     

An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.     

A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade

We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.     

Cloning and characterization of a phospholipase C from the C(4) plant Digitaria sanguinalis

As a PLC activity was implicated in the light transduction pathway that controls C(4) photosynthesis in Digitaria sanguinalis, a full length PLC cDNA (DsPLC2) was cloned. The proteins encoded by the two possible open reading frames were produced in Escherichia coli; they both harbour a PLC activity but with different response to Ca(2+) concentration, and with different sensitivity to the PLC inhibitor U-73122.     

Highly purified intact chloroplasts from mesophyll protoplasts of the C4 plant Digitaria sanguinalis. Inhibition of phosphoglycerate reduction by orthophosphate and by phosphoenolpyruvate

Purified mesophyll protoplasts from the C₄ plant Digitaria sanguinalis were used to prepare intact mesophyll chloroplasts with low cytoplasmic contamination. The procedure involved breakage of protoplasts, differential centrifugation, partition in a dextran-polyethylene glycol two-phase system, and Percoll density gradient centrifugation. The final chloroplast preparation contained about 80% intact chloroplasts with a phosphoenolpyruvate carboxylase contamination of 0.2–1% of the original protoplast activity, corresponding to 1–6 μmol ¹⁴CO₂ fixed/mg Chl h. The purified chloroplasts showed substrate-dependent oxygen evolution in the range of 40–150 μmol substrate reduced/mg Chl h, with phosphoglycerate or oxaloacetate as substrate. Both reactions were stimulated 1.5 fold by pyruvate and further by addition of the other substrate. These measurements indicated that phosphoglycerate reduction was limited by substrate transport across the chloroplast envelope. Without added substrate, the chloroplasts consumed oxygen via pseudo-cyclic electron transport in the light. Also this reaction was stimulated by pyruvate. Phosphoglycerate-dependent oxygen evolution was inhibited by P_i and by phosphoenolpyruvate to about the same extent with purified chloroplasts, but only by P_i with protoplast extracts. This suggests that phosphoglycerate, P_i and phosphoenolpyruvate share a common carrier, similar to the P_i-translocator in C₃ chloroplasts, and that the lack of inhibition obtained with phosphoenolpyruvate and unpurified chloroplasts is artefactual, possibly due to oxaloacetate formation from added phosphoenolpyruvate and concomitant stimulation of oxygen evolution by oxaloacetate reduction. Furthermore, the results suggest that phosphoenolpyruvate is transported with a K_m similar to that of P_i in C₄ mesophyll chloroplasts.     

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.     

C(4) Photosynthesis: Light-dependent CO(2) Fixation by Mesophyll Cells, Protoplasts, and Protoplast Extracts of Digitaria sanguinalis

Mesophyll cells, protoplasts, and protoplast extracts of Digitaria sanguinalis were used for comparative studies of light-dependent CO₂ fixation. CO₂ fixation was low without the addition of organic substrates. Pyruvate, oxaloacetate, and 3-phosphoglycerate induced relatively low rates (10 to 90 μmoles/mg chlorophyll·hr) of CO₂ fixation when added separately. However, a highly synergistic relationship was found between pyruvate + oxaloacetate and pyruvate + 3-phosphoglycerate for inducing light-dependent CO₂ fixation in the mesophyll preparations. Highest rates of CO₂ fixation were obtained with protoplast extracts. Pyruvate, in combination with oxaloacetate or 3-phosphoglycerate induced light-dependent rates from 150 to 380 μmoles of CO₂ fixed/mg chlorophyll·hr which are equivalent to or exceed reported rates of whole leaf photosynthesis in C₄ species. Concentrations of various substrates required to give half-maximum velocities of CO₂ fixation were determined, with the protoplast extracts generally saturating at the lowest substrate concentrations. Chloroplasts separated from protoplast extracts showed little capacity for CO₂ fixation. The results suggest that CO₂ fixation in C₄ mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase.     

The effect of oxygen on CO2 fixation by mesophyll protoplast extracts of C3 and C4 plants.

Oxygen inhibits CO₂ fixation by mesophyll protoplast extracts of the C₃ plants, $\text{Hordeum vulgare}$ and $\text{Triticum aestivum}$, but stimulates the pyruvate induced CO₂ fixation by mesophyll protoplast extracts of the C₄ plants $\text{Digitaria sanguinalis}$ and $\text{Urochloa panicoides}$. The former is reversed by increased levels of bicarbonate, whereas the latter effect is independent of bicarbonate concentration. The results are consistent with the proposal that oxygen inhibits C₃ photosynthesis by competing with CO₂ in the RuDP carboxylase/oxygenase system. The oxygen enhancement of C₄ mesophyll photosynthesis is proposed to be due to pseudocyclic electron flow supplying additional ATP for the CO₂ fixation process.     

Products of CO2 fixation and 14C labelling pattern of alanine in Methanobacterium thermoautotrophicum pulse-labelled with 14CO2

The incorporation of ¹⁴CO₂ by an exponentially growing culture of the autotrophic bacterium Methanobacterium thermoautotrophicum has been studied. The distribution of radioactivity during 2s–120s incubation periods has been analyzed by chromatography and radioautography. After a 2 s incubation most of the radioactivity of the ethanolsoluble fraction was present in the amino acids alanine, glutamate, glutamine and aspartate, whereas phosphorylated compounds were only weakly labelled. The percentage of the total radioactivity fixed, which was contained in the principal early labelled amino acid alanine, increased in the first 20 s and only then decreased, indicating that alanine is derived from primary products of CO₂ fixation.The labelling patterns of alanine produced during various incubation times have been determined by degradation. After a 2 s ¹⁴CO₂ pulse, 61% of the radioactivity was located in C-1, 23% in C-2, and 16% in C-3. The results are consistent with the operation of a previously proposed autotrophic CO₂ assimilation pathway which involves the formation of acetyl CoA from 2 CO₂ via one-carbon unit intermediates, followed by the reductive carboxylation of acetyl CoA to pyruvate.     

Somatic embryogenesis on Thin Cell Layers of a C4 species,Digitaria sanguinalis (L.) Scop

Somatic embryogenesis was obtained from transverse thin cell layers (tTCLs) of Digitaria sanguinalis. tTCLs (0.2 - 0.4mm thick, 1mm in diameter) were excised from 4-week-old seedlings and placed onto Murashige and Skoog media supplemented with a varying concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (from 1 μM to 100 μM) and sucrose (from 3% to 24%). Somatic embryos were obtained in the dark 7-10 days after inoculation from tTCLs excised at specific levels on the seedling and cultured in the presence of 2,4-D (5 μM to 10 μM) and sucrose (3 to 6%). The exposure of the tTCLs to light decreased the percentage of tTCLs forming somatic embryos. Viable plantlets were obtained 2 weeks after transfer onto a cytokinin-containing medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of a nitrogen-fixing bacterial strain from the roots of Digitaria sanguinalis.

Rates of nitrogen fixation of 3 to 10 g of N2 fixed per hectare per day were associated with root systems of Digitaria sanguinalis. A Gram-negative motile aerobic bacterial strain that was capable of N2 fixation was isolated from a washed root sample of one of these plants. Optimal growth and N2 fixation occurred at a pH of about 6.5, a temperature of 30-37 degrees C, and at a pO2 of about 0.01 atm. Increased rates of N2 fixation resulted when this strain was grown in mixed cultures with aerobic or facultative bacteria. Observations of cellular and cultural morphology and results of biochemical and physiological studies indicate that the isolate may be related to the Azotobacteraceae but that it is not identical with any of the members of this family. The importance of N2 fixation by this isolate in nature is unknown.     

14CO2 fixation in incubated rat diaphragms

The time course (0-60 min) of label incorporation from NaH14 CO3 into citric-acid-cycle intermediates and amino acids was investigated in incubations of isolated rat diaphragms. On the basis of these results, 14CO2 exchange by isocitrate dehydrogenase and 14CO2 fixation by propionyl-CoA carboxylation and pyruvate carboxylation could be estimated. Apparent rates amounted to about 30-40, 2, and 35 nmol/min per g of muscle, respectively. About 90 percent of C4-carbon compounds originating from 14CO2 fixation were subsequently removed by decarboxylation. 2-Cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, effectively reduced 14CO2 production from [1-14C]pyruvate but did not affect incorporation of radioactive label from NaH14CO3. In cell-free muscle extracts, 14CO2 fixation was demonstrable under assay conditions suitable for NADP -dependent 'malic' enzyme(s). Addition of hydroxymalonate, an inhibitor of the latter enzyme(s), significantly reduced 14CO2 incorporation. The results provide evidence for a continuous cytosolic replenishment and mitochondrial depletion of citric-acid-cycle carbon skeletons in resting skeletal muscle tissue. The functional role of malic (iso)enzyme activities in these processes is discussed.     

Metagenomic and 14C tracing evidence for autotrophic microbial CO2 fixation in paddy soils

Autotrophic carbon dioxide (CO₂) fixation by microbes is ubiquitous in the environment and potentially contributes to the soil organic carbon (SOC) pool. However, the multiple autotrophic pathways of microbial carbon assimilation and fixation in paddy soils remain poorly characterized. In this study, we combine metagenomic analysis with ¹⁴C-labelling to investigate all known autotrophic pathways and CO₂ assimilation mechanisms in five typical paddy soils from southern China. Marker genes of six autotrophic pathways are detected in all soil samples, which are dominated by the cbbL genes (67%–82%) coding the ribulose-bisphosphate carboxylase large chain in the Calvin cycle. These marker genes are associated with a broad range of phototrophic and chemotrophic genera. Significant amounts of ¹⁴C-CO₂ are assimilated into SOC (74.3–175.8 mg ¹⁴C kg⁻¹) and microbial biomass (5.2–24.1 mg ¹⁴C kg⁻¹) after 45 days incubation, where more than 70% of ¹⁴C-SOC was concentrated in the relatively stable humin fractions. These results show that paddy soil microbes contain the genetic potential for autotrophic carbon fixation spreading over broad taxonomic ranges, and can incorporate atmospheric carbon into organic components, which ultimately contribute to the stable SOC pool.     

Isolation and culture of mesophyll protoplasts from Rosa hybrida

After 1 h plasmolysis in CPW13M solution, highly viable (>75%) protoplasts were isolated from leaves of axenic shoot cultures of Rosa hybrida L. cv. Abraham Darby using an enzyme mixture containing 1.0% (w/v) Hemicellulase, 0.1% (w/v) Macerozyme, 1.0 (w/v) Cellulase RS, 0.05% (w/v) Pectolyase Y23 and 1.0% (w/v) PVP-10 and from cv. Marie Pavié using an identical mixture but with Cellulase RS and Pectolyase Y23 at 0.7% (w/v) and 0.1% (w/v), respectively. With both cvs., sustained protoplast division was achieved after plating in agarose beads with modified KM8p medium containing 1.0% (w/v) polyvinylpyrrolidone (mol. wt. 10 000; PVP-10), 8.91 μM naphthaleneacetic acid (NAA) and 4.44 μM 6-benzyladenine (BA). Protoplast-derived callus gave rise to roots after transfer to SH medium containing 14 μM 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O₂ evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C₄ mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O₂ evolution in the range of 90 to 135 micromoles O₂ per milligram chlorophyll per hour, and up to 180 micromoles O₂ per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O₂ evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K_m (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O₂ evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O₂ evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O₂ evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C₄ mesophyll chloroplast. C₃ spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K_i values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.