S. von Caemmerer

A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species.

Various aspects of the biochemistry of photosynthetic carbon assimilation in C3 plants are integrated into a form compatible with studies of gas exchange in leaves. These aspects include the kinetic properties of ribulose bisphosphate carboxylase-oxygenase; the requirements of the photosynthetic carbon reduction and photorespiratory carbon oxidation cycles for reduced pyridine nucleotides; the dependence of electron transport on photon flux and the presence of a temperature dependent upper limit to electron transport. The measurements of gas exchange with which the model outputs may be compared include those of the temperature and partial pressure of CO2(p(CO2)) dependencies of quantum yield, the variation of compensation point with temperature and partial pressure of O2(p(O2)), the dependence of net CO2 assimilation rate on p(CO2) and irradiance, and the influence of p(CO2) and irradiance on the temperature dependence of assimilation rate.

Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves

A series of experiments is presented investigating short term and long term changes of the nature of the response of rate of CO2 assimilation to intercellular p(CO2). The relationships between CO2 assimilation rate and biochemical components of leaf photosynthesis, such as ribulose-bisphosphate (RuP2) carboxylase-oxygenase activity and electron transport capacity are examined and related to current theory of CO2 assimilation in leaves of C3 species. It was found that the response of the rate of CO2 assimilation to irradiance, partial pressure of O2, p(O2), and temperature was different at low and high intercellular p(CO2), suggesting that CO2 assimilation rate is governed by different processes at low and high intercellular p(CO2). In longer term changes in CO2 assimilation rate, induced by different growth conditions, the initial slope of the response of CO2 assimilation rate to intercellular p(CO2) could be correlated to in vitro measurements of RuP2 carboxylase activity. Also, CO2 assimilation rate at high p(CO2) could be correlated to in vitro measurements of electron transport rate. These results are consistent with the hypothesis that CO2 assimilation rate is limited by the RuP2 saturated rate of the RuP2 carboxylase-oxygenase at low intercellular p(CO2) and by the rate allowed by RuP2 regeneration capacity at high intercellular p(CO2).

Effects of partial defoliation, changes of irradiance during growth, short-term water stress and growth at enhanced p(CO2) on the photosynthetic capacity of leaves of Phaseolus vulgaris L.

The response of CO2-assimilation rate to the intercellular partial pressure of CO2 (p(CO2)) is used to analyse the effects of various growth treatments on the photosynthetic characteristics of P. vulgaris. Partial defoliation caused an increase in CO2-assimilation rate at all intercellular p(CO2). A change in the light regime for growth from high to low light levels caused a decrease of CO2-assimilation rate at all intercellular p(CO2). Growth in a CO2-enriched atmosphere resulted in lowered assimilation assimilation rates compared with controls at comparable intercellular p(CO2). Short-term water stress initially caused only a decline in the CO2-assimilation rate at high intercellular p(CO2), but not at low intercellular p(CO2). Except under severe water stress, changes in the initial slope of the response of CO2-assimilation rate to intercellular p(CO2) were in parallel to those of the in-vitro activity of ribulose-1,5-bisphosphate (RuBP) carboxylase. From the results, we infer that partial defoliation, changes in the light regime for growth, and growth in a CO2-enriched atmosphere cause parallel changes in RuBP-carboxylase (EC 4.1.1.39) activity and the “capacity for RuBP regeneration”, whereas short-term water stress initially causes only a decline in the RuBP-regeneration capacity.

Effect of salinity and humidity on δ13C value of halophytes—Evidence for diffusional isotope fractionation determined by the ratio of intercellular/atmospheric partial pressure of CO2 under different environmental conditions

SummarySeedlings of two mangrove species, Avicennia marina and Aegiceras corniculatum, were grown in a range of salinities and humidities in controlled environment chambers, and Phaseolus vulgaris plants were grown in the glasshouse. The fractionation of carbon isotopes in the three species was correlated with the ratio of intercellular and ambient partial pressures of CO2. The results are consistent with fractionation being due both to diffusion in air and to carboxylation in the leaf. It was concluded that the latter process discriminates against 13CO2 relative to 12CO2 by about 27‰.

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO2. After determination of the rate of CO2 assimilation at known intercellular CO2 pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO2, and fell rapidly as the CO2-assimilation rate increased with increasing intercellular partial pressures of CO2, indicating that bundle-sheath CO2 concentrations fell at low intercellular partial pressures of CO2. In contrast, the amount of phosphoenolpyruvate and of C4-cycle intermediates declined at low intercellular partial pressures of CO2. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C4 cycles.

Simultaneous determination of Rubisco carboxylase and oxygenase kinetic parameters in Triticum aestivum and Zea mays using membrane inlet mass spectrometry

The lack of complete Rubisco kinetic data for numerous species is partly because of the time consuming nature of the multiple methods needed to assay all of the Rubisco parameters. We have developed a membrane inlet mass spectrometer method that simultaneously determines the rate of Rubisco carboxylation (v(c)) and oxygenation (v(o)), and the CO(2) and O(2) concentrations. Using the collected data, the Michaels-Menten equations for v(c) and v(o) in response to changing CO(2) and O(2) concentrations were simultaneously solved for the CO(2) (K(c)) and O(2) (K(o)) constants, the maximum turnover rates of the enzyme for CO(2) (kcat(CO2)) and O(2) (kcat(O2)) and the specificity for CO(2) relative to O(2) (S(c/o)). In the C(4) species Zea mays K(c) was higher but K(o) was lower compared with the C(3) species Triticum aestivum. The kcat(CO2) was higher and the kcat(O2) lower in Z. mays compared with T. aestivum and S(c/o) was similar in the two species. The V(omax)/V(cmax) was lower in Z. mays and thus did not correlate with changes in S(c/o). In conclusion, this mass spectrometer system provides a means of simultaneously determining the important Rubisco kinetic parameters, K(c), K(o), kcat(CO2,)kcat(O2) and S(c/o) from the same set of assays.

Transplastomic integration of a cyanobacterial bicarbonate transporter into tobacco chloroplasts

Improving global yields of agricultural crops is a complex challenge with evidence indicating benefits in productivity are achieved by enhancing photosynthetic carbon assimilation. Towards improving rates of CO2 capture within leaf chloroplasts, this study shows the versatility of plastome transformation for expressing the Synechococcus PCC7002 BicA bicarbonate transporter within tobacco plastids. Fractionation of chloroplast membranes from transplastomic tobBicA lines showed that ~75% of the BicA localized to the thylakoid membranes and ~25% to the chloroplast envelope. BicA levels were highest in young emerging tobBicA leaves (0.12 μmol m–2, ≈7mg m–2) accounting for ~0.1% (w/w) of the leaf protein. In these leaves, the molar amount of BicA was 16-fold lower than the abundant thylakoid photosystem II D1 protein (~1.9 μmol m–2) which was comparable to the 9:1 molar ratio of D1:BicA measured in air-grown Synechococcus PCC7002 cells. The BicA produced had no discernible effect on chloroplast ultrastructure, photosynthetic CO2-assimilation rates, carbon isotope discrimination, or growth of the tobBicA plants, implying that the bicarbonate transporter had little or no activity. These findings demonstrate the utility of plastome transformation for targeting bicarbonate transporter proteins into the chloroplast membranes without impeding growth or plastid ultrastructure. This study establishes the span of experimental measurements required to verify heterologous bicarbonate transporter function and location in chloroplasts and underscores the need for more detailed understanding of BicA structure and function to identify solutions for enabling its activation and operation in leaf chloroplasts.

The effects of Rubisco activase on C4 photosynthesis and metabolism at high temperature

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. T(1) progeny of transgenic Flaveria bidentis (a C(4) dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C(4) photosynthesis at high temperature. A range of T(1) progeny was screened at 25 degrees C and 40 degrees 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 micromol m(-2) (447+/-36 mg m(-2)) compared to a Rubisco site content of 14.2+/-0.8 micromol m(-2). CO(2) assimilation rates and Rubisco carbamylation declined at both 25 degrees C and 40 degrees C when the Rubisco activase content dropped below 3 mumol 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 degrees C compared to 81+/-2% at 25 degrees C. When the CO(2) 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 C(3) and C(4) metabolites pools. It is concluded that during short-term treatment at 40 degrees C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C(4) photosynthesis.

Rubisco Activation is Impaired in Transgenic Tobacco Plants with Reduced Electron Transport Capacity

Rubisco can be catalytically competent only after a specific lysyl residue within the active site has been carbamylated. Before carbamylation can occur, any inhibitory ligands bound at the site must be released, and this process is facilitated by another enzyme, Rubisco activase. It has been shown in vitro that Rubisco activase needs to hydrolyse ATP to function and is inhibited by ADP, and so presumably is sensitive to the chloroplast ATP/ADP ratio (1). However, there are indications that activase is also regulated by transthylakoid pH gradient (∆pH) and electron transport through PSI (2).