Susanne von Caemmerer

Temperature Response of Mesophyll Conductance. Implications for the Determination of Rubisco Enzyme Kinetics and for Limitations to Photosynthesis in Vivo

CO2 transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g m ), is finite. Therefore, it will limit photosynthesis when CO2 is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude ofg m . The process dominatingg m is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO2 in water have all been suggested. The response ofg m to temperature (10°C–40°C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q10) of approximately 2.2 for g m , suggesting control by a protein-facilitated process because the Q10for diffusion of CO2 in water is about 1.25. Further,g m values are maximal at 35°C to 37.5°C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g m to calculate CO2 at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10°C to 40°C. Using these parameters, we determined the limitation imposed on photosynthesis byg m . Despite an exponential rise with temperature, g m does not keep pace with increased capacity for CO2 uptake at the site of Rubisco. The fraction of the total limitations to CO2uptake within the leaf attributable tog m rose from 0.10 at 10°C to 0.22 at 40°C. This shows that transfer of CO2 from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO2 assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO2 partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO2 assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO2, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, kcat, of 3.5+0.2 mol CO2·(mol sites)−1·s−1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO2·(mol sites)−1·−1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO2 and O2, the rate of CO2 assimilation was measured at 25° C at different intercellular partial pressures of CO2 and O2. These measurements were combined with carbon-isotope analysis (13C/12C) of CO2 in the air passing over the leaf to estimate the conductance for transfer of CO2 from the substomatal cavities to the sites of carboxylation (0.3 mol·m−2·s−1·bar−1) and thus the partial pressure of CO2 at the sites of carboxylation. The calculated Michaelis-Menten constants for CO2 and O2 were 259 ±57 μbar (8.6±1.9μM) and 179 mbar (226 μM), respectively, and the effective Michaelis-Menten constant for CO2 in 200 mbar O2 was 549 μbar (18.3 μM). From measurements of the photocompensation point (Γ* = 38.6 ubar) we estimated Rubiscos relative specificity for CO2, as opposed to O2 to be 97.5 in vivo. These values were dependent on the size of the estimated CO2-transfer conductance.

The C4 pathway: An efficient CO2 pump

The C4 pathway is a complex combination of both biochemical and morphological specialisation, which provides an elevation of the CO2 concentration at the site of Rubisco. We review the key parameters necessary to make the C4 pathway function efficiently, focussing on the diffusion of CO2 out of the bundle sheath compartment. Measurements of cell wall thickness show that the thickness of bundle sheath cell walls in C4 species is similar to cell wall thickness of C3 mesophyll cells. Furthermore, NAD-ME type C4 species, which do not have suberin in their bundle sheath cell walls, do not appear to compensate for this with thicker bundle sheath cell walls. Uncertainties in the CO2 diffusion properties of membranes, such as the plasmalemma, choroplast and mitochondrial membranes make it difficult to estimate bundle sheath diffusion resistance from anatomical measurements, but the cytosol itself may account for more than half of the final calculated resistance value for CO2 leakage. We conclude that the location of the site of decarboxylation, its distance from the mesophyll interface and the physical arrangement of chloroplasts and mitochondria in the bundle sheath cell are as important to the efficiency of the process as the properties of the bundle sheath cell wall. Using a mathemathical model of C4 photosynthesis, we also examine the relationship between bundle sheath resistance to CO2 diffusion and the biochemical capacity of the C4 photosynthetic pathway and conclude that bundle sheath resistance to CO2 diffusion must vary with biochemical capacity if the efficiency of the C4 pump is to be maintained. Finally, we construct a mathematical model of single cell C4 photosynthesis in a C3 mesophyll cell and examine the theoretical efficiency of such a C4 photosynthetic CO2 pump.

Factors affecting CO2 assimilation, leaf injury and growth in salt-stressed durum wheat

To examine the factors that affect tolerance to high internal salt concentrations, two tetraploid wheat genotypes that differ in the degree of salt-induced leaf injury (Wollaroi and Line 455) were grown in 150 mM NaCl for 4 weeks. Shoot biomass of both genotypes was substantially reduced by salinity, but genotypic differences appeared only after 3 weeks, when durum cultivar Wollaroi showed greater leaf injury and a greater reduction in biomass than Line 455. Ion accumulation, water relations, chlorophyll fluorescence and gas exchange were followed on one leaf (leaf 3) throughout its life. Salinity caused a large decrease in stomatal conductance (gs) of both genotypes. This was not due to poor water relations, as leaf turgor of both genotypes was higher in the salt treatment than in controls, so chemical signals were likely to have caused the decrease in gs. Reductions in assimilation rate were initially due to gs and, with time, were due to a combination of stomatal and non-stomatal limitations. The non-stomatal limitations were associated with a build up of Na+ above 250 mM. The efficiency of PSII photochemistry in Line 455 was unaffected throughout. However, in Wollaroi, the potential and actual quantum yield of PSII photochemistry began to decline as the leaf aged and the thermal energy dissipation of excess light energy (NPQ) increased. This coincided with high Na+ and Cl- concentrations in the leaf and with chlorophyll degradation, indicating that these later reductions in CO2 assimilation in Wollaroi were a consequence of a direct toxic ion effect. The earlier reduction in CO2 assimilation and greater leaf injury explain why growth of Wollaroi was less than Line 455. The most sensitive indicator of salinity stress was gs, followed by CO2 assimilation, with fluorescence parameters other than NPQ being no more sensitive than chlorophyll itself.

Estimating mesophyll conductance to CO2: methodology, potential errors, and recommendations

The three most commonly used methods for estimating mesophyll conductance (g(m)) are described. They are based on gas exchange measurements either (i) by themselves; (ii) in combination with chlorophyll fluorescence quenching analysis; or (iii) in combination with discrimination against (13)CO(2). To obtain reliable estimates of g(m), the highest possible accuracy of gas exchange is required, particularly when using small leaf chambers. While there may be problems in achieving a high accuracy with leaf chambers that clamp onto a leaf with gaskets, guidelines are provided for making necessary corrections that increase reliability. All methods also rely on models for the calculation of g(m) and are sensitive to variation in the values of the model parameters. The sensitivity to these factors and to measurement error is analysed and ways to obtain the most reliable g(m) values are discussed. Small leaf areas can best be measured using one of the fluorescence methods. When larger leaf areas can be measured in larger chambers, the online isotopic methods are preferred. Using the large CO(2) draw-down provided by big chambers, and the isotopic method, is particularly important when measuring leaves with high g(m) that have a small difference in [CO(2)] between the substomatal cavity and the site of carboxylation in the chloroplast (C(i)-C(c) gradient). However, equipment for the fluorescence methods is more easily accessible. Carbon isotope discrimination can also be measured in recently synthesized carbohydrates, which has its advantages under field conditions when large number of samples must be processed. The curve-fitting method that uses gas exchange measurements only is not preferred and should only be used when no alternative is available. Since all methods have their weaknesses, the use of two methods for the estimation of g(m), which are as independent as possible, is recommended.

Sensitivity of plants to changing atmospheric CO2 concentration : from the geological past to the next century

The rate of CO(2) assimilation by plants is directly influenced by the concentration of CO(2) in the atmosphere, c(a). As an environmental variable, c(a) also has a unique global and historic significance. Although relatively stable and uniform in the short term, global c(a) has varied substantially on the timescale of thousands to millions of years, and currently is increasing at seemingly an unprecedented rate. This may exert profound impacts on both climate and plant function. Here we utilise extensive datasets and models to develop an integrated, multi-scale assessment of the impact of changing c(a) on plant carbon dioxide uptake and water use. We find that, overall, the sensitivity of plants to rising or falling c(a) is qualitatively similar across all scales considered. It is characterised by an adaptive feedback response that tends to maintain 1 - c(i)/c(a), the relative gradient for CO(2) diffusion into the leaf, relatively constant. This is achieved through predictable adjustments to stomatal anatomy and chloroplast biochemistry. Importantly, the long-term response to changing c(a) can be described by simple equations rooted in the formulation of more commonly studied short-term responses.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand

The world’s crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthetic CO2 assimilation

As an approach to understanding the physiological role of chloroplast carbonic anhydrase (CA), this study reports on the production and preliminary physiological characterisation of transgenic tobacco (Nicotiana tabacum L.) plants where chloroplast CA levels have been specifically suppressed with an antisense construct directed against chloroplast CA mRNA. Primary transformants with CA levels as low as 2% of wild-type levels were recovered, together with intermediate plants with CA activities of about 20–50% of wild-type levels. Plants with even the lowest CA levels were not morphologically distinct from the wild-type plants. Segregation analysis of the low-CA character in plants grown from T1 selfed seed indicated that at least one of the low-CA plants appears to have two active inserts and that at least two of the intermediate-CA plants have one active insert. Analysis of CO2 gas exchange of a group of low-CA plants with around 2% levels of CA indicated that this large reduction in chloroplastic CA did not appear to cause a measurable alteration in net CO2 fixation at 350 μbar CO2 and an irradiance of 1000 μmol quanta·m−2·s−1. In addition, no significant differences in Rubisco activity, chlorophyll content, dry weight per unit leaf area, stomatal conductance or the ratio of intercellular to ambient CO2 partial pressure could be detected. However, the carbon isotope compositions of leaf dry matter were significantly lower (0.85%o) for low-CA plants than for wildtype plants. This corresponds to a 15-μbar reduction in the CO2 partial pressure at the sites of carboxylation. The difference, which was confirmed by concurrent measurement of discrimination with gas exchange, would reduce the CO2 assimilation rate by 4.4%, a difference that could not be readily determined by gas-exchange techniques given the inherent variability found in tobacco. A 98% reduction in CA activity dramatically reduced the 18O discrimination in CO2 passing over the leaf, consistent with a marked reduction in the ratio of hydrations to carboxylations. We conclude that a reduction in chloroplastic CA activity of two orders of magnitude does not produce a major limitation on photosynthesis at atmospheric CO2 levels, but that normal activities of the enzyme appear to play a role in facilitated transfer of CO2 within the chloroplast, producing a marginal improvement in the efficiency of photosynthesis in C3 plants.

The Development of C4 Rice: Current Progress and Future Challenges

Another “green revolution” is needed for crop yields to meet demands for food. The international C4 Rice Consortium is working toward introducing a higher-capacity photosynthetic mechanism—the C4 pathway—into rice to increase yield. The goal is to identify the genes necessary to install C4 photosynthesis in rice through different approaches, including genomic and transcriptional sequence comparisons and mutant screening.

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO2 assimilation at known intercellular CO2 pressure, O2 pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O2, photosynthesis appeared RuBP-saturated at low CO2 pressure and RuBP-limited at high CO2 pressure. In 21 mbar (2%) O2, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.