Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.     

C4 Photosynthesis (The CO2-Concentrating Mechanism and Photorespiration)

Despite previous reports of no apparent photorespiration in C4 plants based on measurements of gas exchange under 2 versus 21% O2 at varying [CO2], photosynthesis in maize (Zea mays) shows a dual response to varying [O2]. The maximum rate of photosynthesis in maize is dependent on O2 (approximately 10%). This O2 dependence is not related to stomatal conductance, because measurements were made at constant intercellular CO2 concentration (Ci); it may be linked to respiration or pseudocyclic electron flow. At a given Ci, increasing [O2] above 10% inhibits both the rate of photosynthesis, measured under high light, and the maximum quantum yield, measured under limiting light ([phi]CO2). The dual effect of O2 is masked if measurements are made under only 2 versus 21% O2. The inhibition of both photosynthesis and [phi]CO2 by O2 (measured above 10% O2) with decreasing Ci increases in a very similar manner, characteristically of O2 inhibition due to photorespiration. There is a sharp increase in O2 inhibition when the Ci decreases below 50 [mu]bar of CO2. Also, increasing temperature, which favors photorespiration, causes a decrease in [phi]CO2 under limiting CO2 and 40% O2. By comparing the degree of inhibition of photosynthesis in maize with that in the C3 species wheat (Triticum aestivum) at varying Ci, the effectiveness of C4 photosynthesis in concentrating CO2 in the leaf was evaluated. Under high light, 30[deg]C, and atmospheric levels of CO2 (340 [mu]bar), where there is little inhibition of photosynthesis in maize by O2, the estimated level of CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the bundle sheath compartment was 900 [mu]bar, which is about 3 times higher than the value around Rubisco in mesophyll cells of wheat. A high [CO2] is maintained in the bundle sheath compartment in maize until Ci decreases below approximately 100 [mu]bar. The results from these gas exchange measurements indicate that photorespiration occurs in maize but that the rate is low unless the intercellular [CO2] is severely limited by stress.     

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).     

Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae

As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [¹⁴C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [¹⁴C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[¹⁴C]CABP complex with a combination of polyethylene glycol-4000 and MgCl₂. Plots of the relationship between concentrations of [¹⁴C] CPBP in the reaction mixture and the precipitated [¹⁴C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [¹⁴C]CPBP since the dissociation constant of CABP with Rubisco is 10⁻¹¹ molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C₄ plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one-sixth that of C₃ plant leaves and was affected by the CO₂ concentration during growth. The content of Euglena and blue-green algae is also reported.     

Effects of Elevated Atmospheric CO2 Concentration on Leaf Anatomy and Morphology in Panicum Species Representing Different Photosynthetic Modes

Panicum tricanthum Nees, Panicum antidotale Retz., and Panicum decipiens Nees ex Trin. were selected to represent C3, C4, and C3/C4 intermediate perennial species of Panicum, respectively. Plants grown from seed with 900 ppm [CO2] under natural sunlight and controlled temperatures (30 degrees /22 degrees C) were compared with plants grown with ambient [CO2]. The anatomy of the last fully expanded leaf of the main tiller was studied by light microscopy with computerized graphic image analysis and by transmission electron microscopy. Leaf anatomy did not change qualitatively in response to elevated [CO2], but there were changes in leaf thickness and in the proportions of total transsectional area occupied by mesophyll, bundle sheath cells, vascular elements, and sclerenchyma, according to species. The abaxial stomatal frequency decreased by 22% for P. tricanthum but increased by ca. 30% for the other two species. With 900 ppm CO2, all three species showed a considerable increase in leaf starch content (to >30% of dry matter). Starch granules accumulated in chloroplasts of the mesophyll and bundle sheath cells. Increased leaf glaucousness in response to elevated [CO2] was the result of increased or modified deposition of epicuticular wax on both leaf surfaces, a response to elevated [CO2] that is unusual and one that has not been previously recorded for monocotyledons. The wax patterns were studied by scanning electron microscopy. Panicum decipiens did not respond to elevated [CO2] in a truly intermediate fashion; its responses resembled those of either the C3 or the C4 species. C3/C4 intermediates may thus be interpreted as developmental chimeras and not as species in transition between C3 and C4 modes in an evolutionary sense.     

Photorespiration stoichiometry in leaves estimated by combined physical and stereochemical methods: allowance for isomerase-catalyzed 3H losses in ribulose bisphosphate regeneration

We showed previously [K.R. Hanson and R.B. Peterson (1986) Arch. Biochem. Biophys. 246, 332-346] that under steady-state photosynthetic conditions the fraction of ribulose bisphosphate oxidized and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration) may be estimated in leaves by a combination of physical and stereochemical methods. The calculations assumed that when (3R)-D-[3-3H1,3-14C]glyceric acid is supplied to illuminated leaf discs the only loss of 3H from the combined photosynthetic and photorespiratory system is the result of glycolate oxidase action; i.e., the isomerase-catalyzed losses in the regeneration of ribulose bisphosphate are negligible. The present study of tobacco leaf discs under zero-photorespiration conditions (low O2 and high CO2 concentrations), and also of maize leaf discs, shows that some 3H losses occur (between 8 and 13% of the 3H at C-1 of ribulose 5-phosphate). The calculated loss varied moderately with temperature but did not vary when the flux of ribulose bisphosphate formation was altered by changing the irradiance. The calculated loss under zero-photorespiration conditions, therefore, may be used to calculate ribulose bisphosphate and glycolate partitioning under other conditions. Earlier experiments on the influence of O2 and CO2 concentrations of temperature on the partitioning of ribulose bisphosphate and glycolate have been reexamined. The loss corrections decreased all values for the fraction of ribulose bisphosphate oxidized and increased all values for the stoichiometry of photorespiration. Essentially all stoichiometry values were above the theoretical lower limit of 25%. The previous conclusion that the stoichiometry of photorespiration substantially exceeds 25% at higher O2 concentrations and higher temperatures is unchanged. The results with maize leaf discs implied that there is very little oxidation of ribulose 1,5-bisphosphate under normal-air conditions; i.e., photorespiration is indeed suppressed, not merely hidden, by efficient refixation of CO2.     

Cell position and light influence C4 versus C3 patterns of photosynthetic gene expression in maize.

C4 plants such as maize partition photosynthetic activities in two morphologically distinct cell types, bundle sheath (BS) and mesophyll (M), which lie as concentric layers around veins. We show that both light and cell position relative to veins influence C4 photosynthetic gene expression. A pattern of gene expression characteristic of C3 plants [ribulose bisphosphate carboxylase (RuBPCase) and light-harvesting chlorophyll a/b binding protein in all photosynthetic cells] is observed in leaf-like organs such as husk leaves, which are sparsely vascularized. This pattern of gene expression reflects direct fixation of CO2 in the C3 photosynthetic pathway, as determined by O2 inhibition assays. Light induces a switch from C3-type to C4-type gene expression patterns in all leaves, primarily in cells that are close to a vein. We propose that light causes repression of RuBPCase expression in M cells, by a mechanism associated with the vascular system, and that this is an essential step in the induction of C4 photosynthesis.     

Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts

The response of ribulose 1,5-bisphosphate levels and CO(2) fixation rates in isolated, intact spinach chloroplasts to pyrophosphate, triose phosphates, dl-glyceraldehyde, O(2), catalase, and irradiance during photosynthesis has been studied. Within 1 minute in the light, a rapid accumulation of ribulose bisphosphate was measured in most preparations of intact chloroplasts, and this subsequently dropped as CO(2) fixation increased. Pyrophosphate, triose phosphates, and catalase increased CO(2) fixation and also the levels of ribulose bisphosphate. CO(2) fixation was inhibited by dl-glyceraldehyde and O(2) with corresponding decreases in ribulose bisphosphate. When the rate of photosynthesis decreased at limiting irradiances (low light), the level of ribulose bisphosphate in the chloroplast did not always decrease, suggesting that ribulose bisphosphate was not limiting CO(2) fixation under these conditions. When triose phosphates (fructose bisphosphate plus aldolase) were added to suspensions of chloroplasts at low irradiances, ribulose bisphosphate increased while CO(2) fixation decreased. These observations provide considerable evidence that high ribulose bisphosphate levels clearly are not solely sufficient to permit rapid rates of CO(2) fixation, but that factors other than ribulose bisphosphate concentration are overriding the control of photosynthesis.Isolated chloroplasts are capable of using carbon reserves to produce considerable ribulose bisphosphate. Upon illumination in the absence of CO(2) and O(2), intact chloroplasts produced up to 13 millimolar ribulose bisphosphate.     

CO2 Exchange and Growth of the Crassulacean Acid Metabolism Plant Opuntia ficus-indica under Elevated CO2 in Open-Top Chambers

CO2 uptake, water vapor conductance, and biomass production of Opuntia ficus-indica, a Crassulacean acid metabolism species, were studied at CO2 concentrations of 370, 520, and 720 [mu]L L-1 in open-top chambers during a 23-week period. Nine weeks after planting, daily net CO2 uptake for basal cladodes at 520 and 720 [mu]L L-1 of CO2 was 76 and 98% higher, respectively, than at 370 [mu]L L-1. Eight weeks after daughter cladodes emerged, their daily net CO2 uptake was 35 and 49% higher at 520 and 720 [mu]L L-1 of C02, respectively, than at 370 [mu]L L-1. Daily water-use efficiency was 88% higher under elevated CO2 for basal cladodes and 57% higher for daughter cladodes. The daily net CO2 uptake capacity for basal cladodes increased for 4 weeks after planting and then remained fairly constant, whereas for daughter cladodes, it increased with cladode age, became maximal at 8 to 14 weeks, and then declined. The percentage enhancement in daily net CO2 uptake caused by elevated CO2 was greatest initially for basal cladodes and at 8 to 14 weeks for daughter cladodes. The chlorophyll content per unit fresh weight of chlorenchyma for daughter cladodes at 8 weeks was 19 and 62% lower in 520 and 720 [mu]L L-1 of CO2, respectively, compared with 370 [mu]L L-1. Despite the reduced chlorophyll content, plant biomass production during 23 weeks in 520 and 720 [mu]L L-1 of CO2 was 21 and 55% higher, respectively, than at 370 [mu]L L-1. The root dry weight nearly tripled as the C02 concentration was doubled, causing the root/shoot ratio to increase with CO2 concentration. During the 23-week period, elevated CO2 significantly increased CO2 uptake and biomass production of O. ficus-indica.     

Effects of O2 and CO2 on Nonsteady-State Photosynthesis (Further Evidence for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Limitation)

The effects of CO2 and O2 on nonsteady-state photosynthesis following an increase in photosynthetic photon flux density (PPFD) were examined in Spinacia oleracea to investigate the hypotheses that (a) a slow exponential phase (the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] phase) of nonsteady-state photosynthesis is primarily limited by Rubisco activity and (b) Rubisco activation involves two sequential, light-dependent processes as described in a previous study (I.E. Woodrow, K.A. Mott [1992] Plant Physiol 99: 298-303). Photosynthesis was found to be sensitive to O2 during the Rubisco phase in the approach of photosynthesis to steady state. Analyses of this sensitivity to O2 showed that the control coefficient for Rubisco was approximately equal to 1 during this phase, suggesting that Rubisco was the primary limitation to photosynthesis. O2 had almost no effect on the kinetics (described using a relaxation time, [tau] of the Rubisco phase for leaves starting in darkness or for leaves starting in low PPFD, but [tau] was substantially higher in the former case. CO2 was found to affect both the rate of photosynthesis and the magnitude of [tau] for the Rubisco phase. The [tau] value for the Rubisco phase was found to be negatively correlated with intercellular CO2 concentration (ci), and leaves starting in darkness had higher values of [tau] at any ci than leaves starting in low PPFD. The effects of CO2 and O2 on the Rubisco phase are consistent with the existence of two sequential, light-dependent processes in the activation of Rubisco if neither process is sensitive to O2 and only the second process is sensitive to CO2. The implications of the data for the mechanism of Rubisco activation and for the effects of stomatal conductance on nonsteady-state photosynthesis are discussed.     

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C₄ plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg²⁺ chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used.     

Bundle sheath diffusive resistance to CO(2) and effectiveness of C(4) photosynthesis and refixation of photorespired CO(2) in a C(4) cycle mutant and wild-type Amaranthus edulis

A mutant of the NAD-malic enzyme-type C(4) plant, Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO(2) response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C(4) cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO(2) to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (r(bs)) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate, an inhibitor of PEPC. Values of r(bs) in A. edulis were 70 to 180 m(2) s(-1) mol(-1), increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO(2) to Rubisco in mesophyll cells of C(3) plants. The values of r(bs) in A. edulis are sufficient for C(4) photosynthesis to elevate CO(2) in BS cells and to minimize photorespiration. The calculated CO(2) concentration in BS cells, which is dependent on input of r(bs), was about 2,000 microbar under maximum rates of CO(2) fixation, which is about six times the ambient level of CO(2). High re-assimilation of photorespired CO(2) was demonstrated in both mutant and wild-type plants at limiting CO(2) concentrations, which can be explained by high r(bs). Increasing O(2) from near zero up to ambient levels under low CO(2), resulted in an increase in the gross rate of O(2) evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO(2) in BS cells.     

Mechanism of CO2 acquisition in an acid-tolerant Chlamydomonas

The acid-tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using (14)C-benzoic acid (BA) or [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0-7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO(2) uptake and O(2) evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO(2) uptake. The CO(2) compensation concentration of the cells ranged from 9.4 microM at pH 4.5 to 16.2 microM at pH 7.0. An examination of the kinetics of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the K(m) (CO(2)) was 16.3 microM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO(2), and the expression of external CA at neutral pH values would maintain an equilibrium CO(2) concentration at the cell surface. This species does not possess a CO(2)-concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low K(m) (CO(2)) Rubisco of the alga.     

Measurements of the CO2/O2 specificity of ribulose 1, 5-bisphosphate carboxylase/oxygenase by 31P- and 1H-NMR

High resolution NMR spectroscopy has been demonstrated to be capable of measuring the CO2/O2 specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase. 31P-NMR provides a simple method for quantitatively determining the ratio of the products, 3-phosphoglycerate and phosphoglycolate. Both the specificity factor and degree of the reaction can be measured during the reaction without the need for complete consumption of ribulose bisphosphate or removal of it from the reaction mixture. Inorganic phosphate can also be detected and this may be used for monitoring the phosphatase activity and pH changes. Measurement by highly sensitive 1H-NMR is most time-efficient and is particularly suitable for the Rubisco with a high specificity factor. By optimizing the experimental conditions, it is possible to follow the simultaneous reactions in situ. The NMR method has been applied to three Rubisco enzymes with different values of specificity factor. Both 31P- and 1H-NMR gave similar results, agreeing with those previously reported by other methods.     

Enzyme Co-Localization with Rubisco in Pea Leaf Chloroplasts

Immunocytolocalization experiments indicate that carbonic anhydrase, phosphoribulokinase, and P-glycerate kinase are near neighbors of Rubisco in the pea leaf chloroplast stroma. Direct transfer of ribulose bisphosphate and gaseous CO(2) from phosphoribulokinase and carbonic anhydrase to Rubisco, and direct transfer of P-glycerate from Rubisco to P-glycerate kinase in the chloroplast stroma, is then a possibility. Rubisco activase, responsible for the removal of inhibitory sugar phosphates that bind to the active site of Rubisco in the dark, also appears to be co-localized with Rubisco.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.     

Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle

The limitation to photosynthetic CO2 assimilation in C3 plants in hot, dry environments is dominated by ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) because CO2 availability is restricted and photorespiration is stimulated. Using a combination of genetic engineering and transgenic technology, three approaches to reduce photorespiration have been taken; two of these focused on increasing the carboxylation efficiency of Rubisco either by reducing the oxygenase reaction directly or by manipulating the Rubisco enzyme by concentrating CO2 in the region of Rubisco through the introduction of enzymes of the C4 pathway. The third approach attempted to reduce photorespiration directly by manipulation of enzymes in this pathway. The progress in each of these areas is discussed, and the most promising approaches are highlighted. Under saturating CO2 conditions, Rubisco did not limit photosynthesis, and limitation shifted to ribulose bisphosphate (RuBP) regeneration capacity of the C3 cycle. Transgenic analysis was used to identify the specific enzymes that may be targets for improving carbon fixation, and the way this may be exploited in the high CO2 future is considered.