Abstract Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.
The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T1 progeny of transgenic Flaveria bidentis (a C4 dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C4 photosynthesis at high temperature. A range of T1 progeny was screened at 25 °C and 40 °C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6±0.8 μmol m−2 (447±36 mg m−2) compared to a Rubisco site content of 14.2±0.8 μmol m−2. CO2 assimilation rates and Rubisco carbamylation declined at both 25 °C and 40 °C when the Rubisco activase content dropped below 3 μmol m−2 (125 mg m−2), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44±5% at 40 °C compared to 81±2% at 25 °C. When the CO2 assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C3 and C4 metabolites pools. It is concluded that during short-term treatment at 40 °C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C4 photosynthesis.
C4 plants accumulate more biomass at elevated than at ambient CO2 partial pressure although CO2 assimilation rates are already CO2 saturated at ambient CO2 partial pressures. Ghannoum et al. (2000 PCE 23: 931-942) proposed that reduced stomatal conductance in elevated CO2, might increase leaf temperature which in turn would increase photosynthesis and leaf growth. We tested this hypothesis in a glasshouse experiment with two C4 grasses Bothriochloa bladhii and Astrebla lappacea. Plants were grown in 5 L pots with well watered and nutrient supplemented soil in temperature controlled glasshouse compartments (22/24/26°C in night/morning/day) at three different CO2 partial pressures (120/70/35 Pa, average during the light period). The difference between leaf and air temperature was monitored with 7-12 thermocouples per species and glasshouse over a period of three weeks. Leaf temperature was light dependent reaching highest values at noon (up to 3.8°C in B. bladhii and 2.9°C in A. lappacea when averaged over 10 to 12 plants and 1 hour in 120 Pa CO2). The average leaf-air temperature difference at noon (average of one hour) was for B. bladhii 1.93/1.47/0.97 °C and for A. lappacea 1.53/1.23/0.80 °C at 120/70/35 Pa CO2, respectively. The average leaf-air temperature difference during the full light period was for B. bladhii 1.17/0.89/0.54 °C and for A. lappacea 1.00/0.77/0.52 °C at 120/70/35 Pa CO2 respectively. We conclude, that the leaf temperature is indeed increased with increased CO2 in C4 grasses as we proposed.
Increasing concerns of global climate change have stimulated research interests in all aspects of carbon exchange. This has restored interest in leaf photosynthetic models to predict and assess changes in photosynthetic CO2 assimilation in different environments. This is a comprehensive presentation of the most widely used models of steady-state photosynthesis by an author who is a world authority. Treatments of CO3, CO4 and intermediate pathways of photosynthesis in relation to environment have been update to include work on antisense transgenic plants. It will be a standard reference for the formal analysis of photosynthetic metabolism in vivo by advanced students and researchers.
It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469-477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.
The C4 dicot Flaveria bidentis was genetically transformed with an antisense RNA construct targeted to the nuclear-encoded gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; RbcS). RbcS mRNA levels in leaves of transformants were reduced by as much as 80% compared to wild-type levels, and extractable enzyme activity was reduced by up to 85%. There was no significant effect of transformation with the gene construct on levels of other photosynthetic enzymes. Antisense transformants with reduced Rubisco activity exhibited a stunted phenotype. Rates of photosynthesis were reduced in air at high light and over a range of CO2 concentrations but were unaffected at low light. From these results we conclude that, as is the case in C3 plants, Rubisco activity is a major determinant of photosynthetic flux in C4 plants under high light intensities and air levels of CO2.
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